Characterization of wheat Bell1-type homeobox genes in floral organs of alloplasmic lines with Aegilops crassa cytoplasm

Background Alloplasmic wheat lines with Aegilops crassa cytoplasm often show homeotic conversion of stamens into pistils under long-day conditions. In the pistillody-exhibiting florets, an ectopic ovule is formed within the transformed stamens, and female sterility is also observed because of abnormal integument development. Results In this study, four wheat Bell1-like homeobox (BLH) genes were isolated and named WBLH1 to WBLH4. WBLH1/WBLH3/WBLH4 expression was observed in the basal boundary region of the ovary in both normal pistils and transformed stamens. WBLH2 was also strongly expressed in integuments not only of normal ovules in pistils but also of the ectopic ovules in transformed stamens, and the WBLH2 expression pattern in the sterile pistils seemed to be identical to that in normal ovules of fertile pistils. In addition, WBLH1 and WBLH3 showed interactions with the three wheat KNOX proteins through the BEL domain. WBLH2, however, formed a complex with wheat KNOTTED1 and ROUGH SHEATH1 orthologs through SKY and BEL domains, but not with a wheat LIGULELESS4 ortholog. Conclusions Expression of the four WBLH genes is evident in reproductive organs including pistils and transformed stamens and is independent from female sterility in alloplasmic wheat lines with Ae. crassa cytoplasm. KNOX-BLH interaction was conserved among various plant species, indicating the significance of KNOX-BLH complex formation in wheat developmental processes. The functional features of WBLH2 are likely to be distinct from other BLH gene functions in wheat development.


Background
Alien cytoplasm largely alters gene expression profiles, affecting growth and organogenesis. Nuclear-cytoplasm incompatibility results in abnormal growth phenotypes in higher plants [1,2]. Recurrent backcrossing has been commonly used for production of nuclear-cytoplasmic substitution plants called alloplasmic lines, in which the cytoplasmic genomes are replaced by ones from a related species [3]. Cytoplasmic male sterility is a major phenomenon among the abnormal phenotypes of the alloplasmic lines [1,4]. In many cases of cytoplasmic male sterility, nuclear-cytoplasm incompatibility induces abortion of pollen. Homeotic transformation of stamens into pistil-like structures is sometimes observed in alloplasmic lines of carrot, Brassica napus, tobacco and wheat [5][6][7][8][9], a phenomenon called pistillody.
Cytoplasm of a wild wheat relative, Aegilops crassa, homeotically affects floral organ development and induces pistillody in some alloplasmic common wheat lines lacking fertility restorer genes against Ae. crassa cytoplasm ( Figure 1) [10,11]. The mitochondrial orf260 gene in the Ae. crassa cytoplasm might be associated with induction of the floral homeotic change [12]. An alloplasmic line of the wheat cultivar Norin 26 (N26) with Ae. crassa cytoplasm [(cr)-N26] exhibits male sterility under long-day conditions (> 15 h light period) due to pistillody ( Figure 1E, F), a phenomenon termed photoperiod-sensitive cytoplasmic male sterility [10]. However, an alloplasmic line of the wheat cultivar Chinese Spring (CS) does not because of a fertility restorer gene Rfd1, located on the long arm of chromosome 7B ( Figure 1A) [13]. An alloplasmic line of CS (ditelosomic 7BS) with Ae. crassa cytoplasm [(cr)-CSdt7BS] lacking both long arms of the homologous chromosome 7B shows pistillody unrelated to day length ( Figure 1C), but CS ditelosomic 7BS with the intrinsic cytoplasm (CSdt7BS) forms normal stamens ( Figure 1D) [6].
The Ae. crassa cytoplasm alters expression patterns of wheat class B MADS-box genes at the floral meristem of (cr)-CSdt7BS [6]. Primordia of the pistil-like stamens lack expression of wheat APETALA3 (AP3) and PISTIL-LATA (PI) orthologs such as WAP3, WPI-1 and WPI-2 [14], whereas two wheat class C MADS-box genes, wheat AGAMOUS paralogs (WAG-1 and WAG-2), are ectopically expressed at the pistil-like stamens [15,16]. Despite initiation of ovule formation in these transformed stamens, ectopic ovules incompletely develop, and the transformed stamens are sterile. In addition, pistils are also sterile in the (cr)-CSdt7BS alloplasmic line, and female sterility is due to abnormal ovule development [6]. Both the ovules and ectopic ovules fail to form an inner epidermis and integuments in the chalaza region. This incomplete development of the ovules strongly suggests alteration of gene expression patterns essential to normal ovule formation in pistils and pistillike stamens of the (cr)-CSdt7BS alloplasmic line. However, there is little information about gene expression associated with wheat ovule development.
Following determination of carpel identity via AGA-MOUS (AG), a class C MADS-box gene acting to specify stamen and carpel development in Arabidopsis [17], ovule primordia are initiated within pistils. The initiation of ovule primordium formation is caused by expression of class D MADS-box genes such as Arabidopsis SEEDSTICK (STK) and petunia floral binding protein 7 (FBP7) and FBP11 [18][19][20]. ARABIDOPSIS BSISTER (ABS) and FBP24 from petunia are members of the B sister subfamily, and are necessary to determine the identity of the endothelial layer within the inner integument of the ovule [21,22]. Our previous study using wheat alloplasmic lines with Ae. crassa cytoplasm demonstrated that alteration of class B and C MADS-box genes was Figure 1 Photoperiod-sensitive cytoplasmic male sterility in alloplasmic wheat with Ae. crassa cytoplasm. Genetic characteristics of nuclear and cytoplasmic types and floral organ morphology without the lemma in the alloplasmic lines are schematically represented. (A) (cr)-CS has normal stamens because of the fertility restorer gene Rfd1 against the Ae. crassa cytoplasm on the long arm of chromosome 7B. (B) (cr)-CS monotelodisomic 7BS, which has Rfd1 in a hemizygous state, shows partial pistillody (arrow) and reduced female fertility. (C) (cr)-CS ditelosomic 7BS, which lacks both of the long arms pair of chromosome 7B and therefore lacks Rfd1, shows complete pistillody and female sterility, independent of day length. Floral organ morphology of CS ditelosomic 7BS was indistinguishable from that of CS. (D) Euplasmic CS. (E) (cr)-N26 shows homeotic transformation of stamens into pistils (pistillody) only under long-day conditions. (F) (cr)-N26 occasionally shows partial pistillody (arrow) and reduced female fertility under short-day conditions. (G) An EMS-induced mutant of (cr)-N26 shows normal floral phenotype [36]. This mutant fails to measure day length. (H) (cr)-N61, which contains multiple loci for Rfd genes, shows normal floral phenotype. Pa, palea; P, pistil; S, stamen; L, lodicule; pTs, partially transformed stamen; Ts, transformed stamen.
connected with continuous transcript accumulation of class D and B sister MADS-box genes, WSTK and WBsis, respectively, in primordia of ectopic ovules within the pistil-like stamens [23]. Moreover, the WBsis transcript is not restricted to the endothelium and appears at the nucellus in ectopic ovules in (cr)-CSdt7BS. Arabidopsis AINTEGUMENTA (ANT) plays an important role in ovule development along the proximal-distal axis [24]. ant mutants fail to both initiate and elongate the integument [25,26]. The aberrant ovule formation is at least partly associated with the weak expression of a wheat ANT homolog WANT-1 around ovule primordia in (cr)-CSdt7BS [16]. Therefore, alteration of gene expression after initiation of ovule primordia results in abnormal ovule development in alloplasmic wheat with Ae. crassa cytoplasm.
Arabidopsis homeodomain protein BELL1 (BEL1) is also required for ovule morphogenesis [27], suggesting that wheat BEL1 homologs may be associated with ovule development. BEL1 acts to specify integument identity by controlling AG activity [28]. BEL1 encodes a TALE homeodomain transcription factor distantly related to the PBX family [27]. Plant BEL1-like homeobox (BLH) genes form a small gene family functioning in various developmental aspects such as seed shattering in rice [29] and leaf shape establishment in Arabidopsis [30]. For many of the BLH functions, molecular interaction with KNOTTED1-type homeodomain (KNOX) proteins is required. Barley JuBel1 and JuBel2 were originally isolated as interaction partners of the KNOX protein BKN3 [31]. Arabidopsis BELLRINGER (syn. PENNYWISE) functions with KNOX genes in early patterning of inflorescence architecture and in maintenance of the inflorescence meristem [32,33]. The BEL domain of BLH directly interacts with the MEINOX domain of KNOX, and the interaction is evolutionarily conserved between animals and plants [34,35].
To elucidate molecular mechanisms of the abnormal ovule development in alloplasmic wheat with Ae. crassa cytoplasm, we first isolated wheat cDNA clones for the BLH homologs, and then studied their differential expression patterns and interaction with wheat KNOX proteins. Association of the BLH homologs with pistillody and female sterility in (cr)-CSdt7BS is discussed based on the results.

Plant materials
Three common wheat (Triticum aestivum L., genome constitution AABBDD) cultivars, Chinese Spring (CS), Norin26 (N26) and Norin61 (N61), and a ditelosomic 7BS line of CS (CSdt7BS) were used in this study. Euplasmic lines of each with their intrinsic cytoplasm develop normal fertile reproductive organs. Alloplasmic lines with the cytoplasm of a wild relative, Aegilops crassa, used in the study were respectively abbreviated as (cr)-CS, (cr)-N26, (cr)-N61 and (cr)-CSdt7BS. The (cr)-N26 shows pistillody when grown under long-day conditions ( Figure 1E), and exhibits partial pistillody under short-day conditions, implying that N26 might contain a fertility restorer (Rf) gene functioning under short-day conditions ( Figure 1F) [10]. Both (cr)-CS and (cr)-N61 show normal male fertility because of their possession of Rf gene(s) against the Ae. crassa cytoplasm in their nuclear genome ( Figure 1A, H) [11]. CS has a single dominant Rf gene, Rfd1, on the long arm of chromosome 7B (7BL) and N61 has multiple Rf genes. A CS monotelodisomic line of chromosome 7BS with the Ae. crassa cytoplasm [(cr)-CSmt7BS] is hemizygous for Rfd1 and partially fertile ( Figure 1B). (cr)-CSdt7BS, which was generated from a cross of (cr)-CSmt7BS with CSdt7BS, shows not only pistillody but also female sterility, independently of day length ( Figure 1C) [6]. An Fr mutant of (cr)-N26 obtained after EMS mutagenesis of (cr)-N26 shows no pistillody even under long-day conditions ( Figure 1G) [36], and was used in the expression analysis.
Three accessions of ancestral diploid species, Triticum urartu (AA), Aegilops speltoides (SS) and Aegilops tauschii (DD), and the tetraploid wheat (Triticum durum, AABB) cultivar Langdon were used in DNA gel blot analysis. A nulli-tetrasomic series of CS produced by Sears [37] was used for chromosome assignment of the isolated wheat cDNAs. Each line of the nulli-tetrasomic series lacks a given pair of homoeologous A, B or D genome chromosomes (the nullisomic condition) that have been replaced by the corresponding homoeologous chromosome pair (the tetrasomic condition).
Cloning and sequencing of wheat BELL1 homologs Degenerate primers, 5'-CGA(A/G)CACTTCCT(A/G/C/T) CACCCGT-3' and 5'-AC(C/G)C(G/T)(C/G/T)GCGTTGAT (A/G)AACCA-3', were designed and used for amplification of the BEL domain regions in RNA from pistils of CS and (cr)-CSdt7BS. The reverse transcription (RT)-PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and nucleotide sequences were determined by an automated fluorescent DyeDeoxy terminator cycle sequencing system using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences of the isolated cDNA fragments were analyzed by DNASIS software (Hitachi, Tokyo, Japan) and the sequence was searched for homology using the BLAST algorithm [38].

Southern blot and RT-PCR analyses
For genomic Southern blot analysis, total DNA extracted from CS, Langdon and diploid wheat was digested with the restriction enzyme HindIII. Total DNA extracted from the nulli-tetrasomics was also digested with HindIII, DraI and BamHI. The digested DNA was fractionated by electrophoresis through a 0.8% agarose gel, transferred to Hybond N + nylon membrane (GE Healthcare, Piscataway, NJ, USA) and hybridized with 32 P-labeled partial BLH cDNA fragments as a probe ( Figure 2). Probe labeling, hybridization, washing and autoradiography were performed according to Takumi et al. [40].
Quantitative RT-PCR was performed using a TaKaRa Thermal Cycler Dice Real Time System (TaKaRa Bio, Ohtsu, Japan) and gene-specific primer sets. As an endogenous control, the wheat Act gene was used. The rate of amplification was monitored using THUNDERBIRD SYBR pPCR Mix (Toyobo, Osaka, Japan) according to In addition to WBLH1 to WBLH4, six rice BLH, eight Arabidopsis BLH and barley JUBEL1 and JUBEL2 proteins were included. Homeodomain sequences of WKNOX1, which is a member of the wheat class 1 KNOX protein family, and Caenorhabditis elegans CEH-20, which is a homolog of PBX protein, were used as outgroups. The phylogenetic tree was constructed according to the UPGMA method. the manufacturer's protocol. Results were obtained as 2 -Δ Ct values, where Ct is the number of PCR cycles required to reach the log phase of amplification for the examined genes minus the same measure for Act, and were represented as values relative to the transcript levels in pistils of CS, N61 or CSdt7BS.

mRNA in situ hybridization
Pistils and young spikes at the stage of floral organ development in the lines CSdt7BS and (cr)-CSdt7BS were fixed in 3.7% p-formaldehyde, 5% acetic acid at 4°C overnight. The fixed tissues were embedded in Paraplast medium (Oxford Labware, St. Louis, MO, USA) after dehydration with ethanol and infiltration with xylene. The embedded samples were sliced into 8-μm sections and dried overnight onto slides coated with VECTA-BOND Reagent (Vector Laboratories, Burlingame, CA, USA). Tissue sections were deparaffinized with xylene and hydrated through an ethanol series, then dehydrated through an ethanol series after treatment with proteinase K and triethanolamine. Hybridization of the four BLH mRNAs with digoxigenin-labeled probe produced from the coding regions was performed overnight at 52°C. The RNA probes were directly synthesized with T3 and T7 RNA polymerase (Toyobo) from the isolated cDNA clone. After hybridization, the sections were washed and treated with RNase. Immunological detection of the hybridized probe was according to Morimoto et al. [42].

Results
Cloning of wheat Bell1-like homeobox genes The CS line ditelosomic 7BS lacking Rfd1 with normal cytoplasm (CSdt7BS) forms normal stamens, whereas the alloplasmic line of CSdt7BS with Ae. crassa cytoplasm ((cr)-CSdt7BS) showed pistillody in all florets ( Figure 1). To identify wheat BLH homologs associated with pistillody, we designed degenerate primers based on the nucleotide sequences of rice and barley BLH homologs, and amplified the 108-bp regions encoding the conserved BELL1-type homeodomains using total RNA from pistils of CS and (cr)-CSdt7BS. In total, 63 of the RT-PCR products were sequenced and classified into seven groups based on their nucleotide sequences (Additional file 1), indicating that at least seven members of the BLH gene family were identified in the wheat genome. Four of the identified BLH members, groups 1, 2, 3 and 7, appeared favored by either CS or (cr)-CSdt7BS.
In the wheat EST database of the Cereal Research Centre, Agriculture and Agri-Food Canada, one EST clone showed high homology to barley JuBel1 [31], and the cDNA clone TaLr1107F03R contained an entire ORF. The wheat JuBel1 homolog corresponding to the group 3 RT-PCR products was named WBLH1. Two additional cDNA sequences for WBLH1 were isolated from CS. The three WBLH1 cDNAs contained single ORFs encoding 767, 765 and 771 amino acid residues, and were respectively designated WBLH1-1, WBLH1-2, and WBLH1-3. To isolate full-length cDNA clones for the group 1, 2 and 7 RT-PCR products, 5'-and 3'-RACE-PCR were conducted with gene-specific primers designed for the homeobox regions. Based on the nucleotide sequences of the RACE-PCR products, three cDNA-specific primer sets were designed, and the cDNA clones identified were named WBLH2, WBLH3 and WBLH4. A WBLH2 cDNA clone encoding 547 amino acid residues was obtained, and the cDNA sequence included an 18-bp deletion in the ORF compared with the RACE-PCR product. The WBLH3 and WBLH4 cDNA clones contained an entire ORF encoding 580 and 623 amino acids, respectively. The isolated cDNA sequences were deposited in the DDBJ database under the accession numbers AB546641-AB546647.
Putative amino acid sequences of the four wheat BLH proteins contained three conserved domains, SKY, BEL and homeodomains (Figure 2A). Based on the homeodomain sequences of four WBLH and related proteins, a phylogenetic tree was constructed ( Figure 2B). The amino acid sequence of WBLH1-1 showed the highest similarity (90.2% identity) to that of barley JuBEL1, and JuBEL2 was closely related (78.7% identity) to WBLH4. WBLH3 showed high similarity to rice qSH1, a major quantitative trait locus controlling seed shattering through formation of an abscission layer [29]. The homeodomain of WBLH2 was highly similar to that of Arabidopsis light-induced ATH1 [46] and distantly divergent from other BEL1-related proteins. In all three conserved domains, WBLH1 showed the highest similarity to Arabidopsis BEL1 of the four WBLH proteins. Among these WBLH proteins, homeodomain sequences were highly conserved, while the SKY and BEL domains were relatively more diverged (Additional file 2). In particular, the SKY and BEL domains of WBLH2 were distinct from those of the other three WBLH proteins.
To study the copy number of WBLH1 in the wheat genome, Southern blots were analyzed using total DNA isolated from diploid, tetraploid and hexaploid wheat. Southern blots using the shorter cDNA fragment as a probe (Figure 2A) showed low copy numbers of WBLH1 in tetraploid and hexaploid wheat genomes, and a single major and a few minor bands for WBLH1 in the A, S and D diploid genomes (Additional file 3). To assign the WBLH1 homologous loci to wheat chromosomes, aneuploid analysis was performed using a series of nullitetrasomic lines. Southern blots using the longer cDNA fragment as a probe (Figure 2A) showed that WBLH1homologous bands could be assigned to chromosomes 2B, 4A, 4D, 5A, 5B and 5D (Additional file 3), meaning that the three isolated WBLH1 cDNAs were not necessarily homoeologous. Two major bands of WBLH2 were observed in the A, S and D diploid genomes (data not shown), and the WBLH2-specific bands were absent only in the nulli-tetrasomic lines of homoeologous group 7 chromosomes and chromosomes 1A, 2A and 2D (Additional file 3). WBLH2 and its homologous loci were assigned to the six chromosomes in common wheat. Similarly, a single major band for WBLH3 and WBLH4 was detected in the A, S and D diploid genomes (data not shown). The WBLH3-and WBLH4specific bands were absent in the nulli-tetrasomic lines of homoeologous group 1 and 3 chromosomes, respectively (Additional file 3). These Southern bots indicated that WBLH3 and WBLH4 respectively represented three homoeologous loci of group 1 and 3 chromosomes in common wheat.

Expression patterns of the four WBLH genes revealed by RT-PCR analyses
To study expression patterns of the four WBLH genes in wheat, RT-PCR analysis was conducting using total RNA from various tissues of CS. Single bands were clearly visualized for the four WBLH genes, although the gene-specific primers did not distinguish the three A, B, and D homoeoalleles of the target genes. The four wheat BLH genes showed tissue-specific expression patterns ( Figure 3). WBLH1 transcripts accumulated predominantly in floral organs than in vegetative organs, suggesting that these genes play important roles in wheat reproductive organ development. WBLH2 transcripts were detected in shoot and inflorescence meristem-containing tissues, and in floral organs. WBLH3 expression was observed in most tissues examined, and the expression pattern of WBLH4 closely resembled that of WBLH3.
Next, to compare expression patterns of the four WBLH genes in wheat florets, semi-quantitative and quantitative RT-PCR analyses were conducted using total RNA isolated from floral organs of various alloplasmic wheat lines with Ae. crassa cytoplasm. WBLH1 Figure 3 RT-PCR analysis of four BLH and three KNOX genes in the normal line (CS). Total RNA was isolated from roots, embryos on day 1 of germination, leaf blades, leaf sheaths, ligules and auricle-containing leaf regions, glumes, paleae, lemmas, stamens, pistils, young spikes (15-25 mm in length) and immature embryos 10 days after anthesis. The Act gene was used as an internal control. Number of PCR cycles is shown on the right of each panel.
transcripts were more abundantly accumulated in stamens than in pistils and lodicules of CS, and were observed at a low level in leaves of CS seedlings ( Figure  4). Similarly, abundant accumulation of WBLH1 transcripts was observed in partially transformed stamens of (cr)-CSmt7BS, but no significant difference in the WBLH1 transcript level was observed between stamens of (cr)-CS and the partially transformed stamens of (cr)-CSmt7BS ( Figure 5). In transformed stamens of (cr)-CSdt7BS, the transcript level was similar to that in the pistils of both euplasmic and alloplasmic CS lines. To confirm abundant expression in stamens, WBLH1 expression was also analyzed using the other series of euplasmic and alloplasmic wheat lines. The WBLH1 transcripts similarly accumulated in stamens of all the lines examined ( Figure 4). In partially transformed stamens of (cr)-N26, the WBLH1 transcript level was significantly reduced compared with euplasmic N26 ( Figure 5).
Abundant accumulation of WBLH2 transcripts was observed in pistils and lodicules, with no transcript detected in leaves and roots of CS (Figure 4). The abundant expression of WBLH2 in pistils was also observed in the other euplasmic and alloplasmic lines. The WBLH2 transcript levels in transformed stamens of (cr)-CSdt7BS and partially transformed stamens of (cr)-CSmt7BS and (cr)-N26 were significantly reduced compared with those in pistils, whereas transformed stamens of (cr)-CSdt7BS had an increased WBLH2 transcript level compared with normal stamens of CSdt7BS ( Figure 5). The WBLH2 transcripts in the partially transformed stamens of (cr)-CSmt7BS and (cr)-N26 accumulated at lower levels than those of fully transformed stamens.
WBLH3 and WBLH4 transcripts more abundantly accumulated in pistils and lodicules compared with stamens of CS (Figure 4). WBLH3 expression was also detected in leaves and roots. WBLH4 transcript levels were more abundant in the lodicules than in the pistils. In pistils of the other euplasmic and alloplasmic lines, predominant expression of WBLH3 and WBLH4 was clearly observed. The WBLH3 and WBLH4 transcript levels in transformed stamens of (cr)-CSdt7BS were similar to those in pistils.
Wknox1 is an ortholog of the maize kn1 homeobox gene functioning mainly in shoot, inflorescence and floral meristem [40,42]. WRS1 and WLG4 are wheat orthologs of maize rough sheath1 (rs1) and liguleless4 (lg4) KNOX genes, respectively [43,44]. Maize rs1 and lg4 belong to the class I KNOX gene family and play important roles in maintenance of shoot apical meristem (SAM) indeterminancy and differentiation of lateral organs [47][48][49]. Transcripts of Wknox1, WRS1 and WLG4 accumulated abundantly in SAM-containing embryos and young spikes and in floral organs, but not in fully developed leaf blades (Figure 3). Accumulation of Wknox1 transcript was observed in pistils and lodicules of CS ( Figure 4). Low levels of Wknox1 transcripts were detected in stamens of euplasmic and alloplasmic lines of CS, whereas slight Wknox1 transcript accumulated in partially transformed stamens of (cr)-CSmt7BS. Wknox1 transcripts accumulated abundantly in transformed stamens of (cr)-CSdt7BS, and the accumulation level was similar to that in pistils. Abundant expression of Wknox1 in pistils was also observed in the other euplasmic and alloplasmic lines.

In situ localization of the four WBLH transcripts in pistils
Fully developed ovules in normal pistils are surrounded by the inner and outer integuments, all enveloped by an inner epidermis ( Figure 6A) [23]. To compare the expression patterns of the four WBLH genes in pistil development of common wheat in detail, in situ hybridization analysis was conducted using pistils and young spikes at the floral organ developing stage, in which stamen and pistil primordia develop. In transverse sections of pistils from CS and CSdt7BS, accumulation of WBLH1 transcript was found in the ovary, but not the ovule ( Figure 6B). In both pistils and transformed stamens of (cr)-CSdt7BS, WBLH1 transcripts were localized to the ovary but not the ovule. No significant differences in WBLH1 localization were observed among fertile pistils, sterile pistils, and transformed stamens. WBLH1 transcripts were also found in longitudinal sections of anthers, especially in tapetum and anther epidermis of CS ( Figure 6C).
WBLH2 transcripts highly accumulated at the stage of floral organ development in the ovule of the CS, CSdt7BS and (cr)-CSdt7BS pistils ( Figure 6D). WBLH2 mRNA was present at higher levels in the inner integument than in the outer integument and nucellus of the pistils. No significant difference in WBLH2 mRNA localization was observed between fertile and sterile pistils. Even in ectopic ovules of the transformed stamens, WBLH2 transcripts highly accumulated, although the integument developed incompletely.
In longitudinal sections of young spikes at the floral organ developing stage from CSdt7BS, WBLH3 mRNA was detected in the anther walls of the stamen and the basal region of the carpel but not in the ovule ( Figure  6E). WBLH3 transcripts accumulated in the central region of the transformed stamens of (cr)-CSdt7BS, where the ectopic ovule was presumed to develop. Similar expression patterns were observed for WBLH4 (data not shown).

Protein-protein interaction between wheat BLH and KNOX
Protein-protein interactions between BLH and KNOX were previously reported in Arabidopsis and barley [31,34]. The BLH-KNOX heterodimers play important roles in plant development [30,32,33,50]. To confirm the interactions between wheat BLH and KNOX proteins, a yeast two-hybrid assay was conducted. Entire ORF sequences of Wknox1, WRS1 and WLG4 were fused to the nucleotide sequence for the yeast GAL4 DNA binding domain (BD-WKNOX1, BD-WRS1 and BD-WLG4), and the WBLH1, WBLH2 and WBLH3 ORFs were fused to the sequence for the yeast GAL4 transcriptional activation domain (AD-WBLH1, AD-WBLH2 and AD-WBLH3). The yeast two-hybrid assay indicated that WBLH1 interacts with WKNOX1, WRS1 and WLG4 (Figure 7). WBLH3 also showed interaction with the three wheat KNOX proteins. WBLH2, however, formed a complex with WKNOX1 and WRS1, but not with WLG4.
To determine the KNOX-interacting domain of wheat BLH proteins, deletion-mutant constructs of WBLH1 and WBLH2 were produced, and protein-protein interaction of the WBLH1 and WBLH2 deletion proteins with WKNOX1 and WLG4 was examined by yeast twohybrid assay (Figure 8). NoWBLH1 deletion construct lacking the BEL domain showed positive interaction with WKNOX1 and WLG4 ( Figure 8A). Neither Figure 5 Quantitative RT-PCR analysis of WBLH1 and WBLH2 in euplasmic and alloplasmic lines of CS. Act was used as an internal control. The transcript levels are shown as values relative to the mRNA levels in pistils of CS. Data are represented as means with standard deviation (n = 3 or n = 4). One and two asterisks respectively indicate statistical significance between pistil and stamen at the 5% and 1% levels (Student's t test). P, pistil; S, stamen; pTs, partially transformed stamen; tSt, transformed stamen. deletion in the SKY domain nor the homeodomain of WBLH1 had any effect on protein-protein interactions with the two KNOX proteins. The N-terminal region of WBLH1 was also not required for the interactions. Arabidopsis BEL1 interacts with KNOX proteins through the N-terminal to homeodomain regions [34]. The interaction domain with KNOX was more restricted in WBLH1 than in BEL1. Similarly, deletions in the SKY and BEL domain region of WBLH2 abolished the interaction with WKNOX1, and a WBLH2 deletion without the homeodomain had no influence on the interaction ( Figure 8B). Deletion analysis of WBLH2 indicated that the region from the SKY domain to the BEL domain was required for the interaction with WKNOX1. In addition, no deletion derivatives of WBLH2 showed positive interactions with WLG4, meaning the absence of any region inhibiting the interaction with WLG4 in WBLH2.
The entire ORF of WBLH1 was fused to the nucleotide sequence for the yeast GAL4 DNA binding domain. Yeast transformants carrying the BD-WBLH1 construct survived on Hisselection medium, indicating that WBLH1 could activate transcription of the reporter gene used in yeast. To identify the activation-related domain, deletion derivatives of WBLH1 fused to the DNA binding domain were introduced, and transformants were recovered on Hisselection medium. No WBLH1 constructs lacking either the SKY, BEL or homeodomains lost function as a transcriptional activator ( Figure 8A). Deletion of the N-terminal region failed to activate reporter gene expression. These results revealed that the N-terminal region (119th to 349th amino acid residues) was essential for transcriptional activation.

Discussion
WBLH2 function is distinct from those of other three BLH genes Bell1-type homeobox genes play important roles in plant development. In this study, four wheat BLH genes were isolated and named WBLH1 to WBLH4. WBLH1, a putative ortholog of barley JuBEL1 [31], was highly similar to Arabidopsis SAWTOOTH1 (SAW1) and SAW-TOOTH2 (SAW2), which act redundantly to suppress expression of the KNOX gene BREVIPEDICELLUS (BP) in leaf margins [30]. SAW1 and SAW2 expression was also observed in lateral organs including the adaxial side of developing sepals but not in developing ovules, and therefore SAW1 and SAW2 are unlikely to be redundant with BEL1 function in ovule development, although the two genes are the most closely related in sequence to BEL1 [30]. WBLH1 was expressed in the ovary but not the ovule ( Figure 6B), indicating that WBLH1 function also does not correspond to that of BEL1.
Both BEL1 and SAW1 are able to interact with class I KNOX proteins, STM, BP and KNAT2, and with the class II KNOX protein KNAT5, but not with other class II KNOX proteins, KNAT3, KNAT4 and KNAT7 [30,35]. The class I KNOX genes are considered to play a role in establishment and maintenance of meristematic identity and in the initiation of lateral organ primordia, and their expression patterns were temporally and spatially regulated, whereas expression of the class II KNOX genes is constitutive [47,51,52]. Similarly, WBLH1 could interact with three class I KNOX proteins, WKNOX1, WRS1 and WLG4 (Figure 7). Wknox1, WRS1 and WLG4 are related to KNAT1, BP and KNAT5, respectively [36,40,43]. Barley JuBEL1 also interacts with class I KNOX proteins BKN1 and BKN3, but not with class II KNOX protein BKN7 [31]. The interacting domain of WBLH1 could be defined to the BEL domain in this study (Figure 8), which was included in the interacting region of JuBEL1 [31]. These results imply evolutionary conservation of the protein-protein interaction between the class I KNOX proteins and WBLH1-related proteins since the divergence of monocotyledonous and dicotyledonous plants. In animals, interaction between two types of TALE-homeobox protein families plays important roles in development, and an N-terminal portion of the PBX subclass proteins is essential for heterodimerization with the MEINOX subclass proteins [53,54]. The MEINOX-PBX complex in animals is evolutionarily conserved as the KNOX-BLH  complex in plants [34]. KNOX-BLH interaction likely alters intracellular localization of the KNOX transcription factor [55], and the KN1-BLH complex binds to the specific DNA motif, TGACAG(G/C)T, with higher affinity compared with KN1 alone [56]. These observations surely indicate the significance of KNOX-BLH complex formation in various plant developmental processes.
WBLH3 also interacted with the three class I KNOX proteins (Figure 7). WBLH3 was phylogenetically close to WBLH4, a putative ortholog of barley JuBEL2 [31,57], although the chromosomal locations of WBLH3 and WBLH4 were different. RT-PCR and in situ mRNA hybridization analyses revealed that the gene expression profile of WBLH3 was generally identical to that of WBLH4. WBLH3 and WBLH4 were homologous to Arabidopsis BLH1 ( Figure 2B). WBLH3 and WBLH4 expression was observed in the basal region of the carpel but not the ovule ( Figure 6E). Similarly, Arabidopsis BLH1 expression is restricted to the transmitting tract and the base of the funiculus, but is not observed in the ovule or embryo sac [58]. Therefore, the WBLH3/4 expression patterns were well conserved with that of BLH1. Misexpression of BLH1 affects normal embryo sac development in ovules of the Arabidopsis eostre mutant [58]. A mutation of the class II KNOX gene KNAT3 suppresses the eostre mutant phenotype [58], and KNAT3 is able to interact with BLH1 [58]. Hackbusch et al. [59] and Pagnussat et al. [58] also reported that the function of the Arabidopsis KNOX-BLH complexes requires the ovate family proteins. No misexpression of WBLH3/4 was observed in ovules of the euplasmic and alloplasmic wheat lines, and therefore we have no information about functional orthology between BLH1 and WBLH3/ 4. WBLH3 displays similarity to the rice qSH1 homeobox protein, a putative ortholog of the Arabidopsis REPLUMLESS (RPL) homeobox protein [29,60]. RPL and qSH1 are associated with seed shattering. RPL is expressed in Arabidopsis stem, pedicels and replum of developing ovaries, and qSH1 is expressed in rice inflorescence meristem, anther and the boundary of the spikelets. WBLH3 might function to define the boundary regions as do RPL and qSH1.
WBLH2, closely related to the Arabidopsis ATH1 homeobox gene, was phylogenetically distinct from other wheat BLH genes ( Figure 2B). ATH1 was originally isolated as a light-induced homeobox gene [46]. ATH1 is expressed in shoot, inflorescence and floral meristems, developing stamens and carpels, and basal regions of lateral organs including leaves, sepals and petals [61]. The mutant phenotype of ath1 indicates that ATH1 controls the development of the boundary region between shoot lateral organs and the stem [61]. The ATH1-STM complex is associated with initiation and maintenance of Arabidopsis SAM [50]. AHT1 expression in stamens and carpels is consistent with the activation of ATH1 by AGAMOUS [62]. In contrast to the ATH1 expression pattern, WBLH2 was abundantly expressed in ovules but not in ligule or auricle regions (Figure 3, Figure 6D). The WBLH2 expression pattern is likely to be analogous to that of BEL1 rather than ATH1. Especially in integuments of ovules, WBLH2 transcripts accumulated abundantly, indicating that WBLH2 might be associated with development of integuments in wheat. The ovule-specific expression of WBLH2 clearly differed from the expression patterns of three other wheat BLH genes. In addition, of the class I KNOX proteins examined, WBLH2 was not able to interact with WLG4 ( Figure 7). The interacting region of WBLH2 with WKNOX1 was defined from the SKY domain to the BEL domain, which was broader than the interacting region of WBLH1 with WKNOX1 and WLG4 ( Figure 8). These observations reveal that the functional features of WBLH2 are distinct from those of the other BLH gene functions in wheat development. In particular, the role of WBLH2 in ovule development should be elucidated in further studies.

Altered expression of BEL1-type homeobox genes in floral organs of alloplasmic lines
Pistillody is one of the typical phenomena appearing in nuclear-cytoplasm incompatibility of higher plants. For expression of pistillody, the expression patterns of a large number of nuclear genes including class B MADSbox genes are altered [6,63]. The downregulation of the class B MADS-box genes in stamen primordia results in homeotic conversion at floral whorl three in the pistillody-showing alloplasmic lines as well as loss-of-function mutants of the class B MADS-box genes. Within their pistil-like stamens, ectopic ovules are surely formed as reported in Arabidopsis, rice and alloplasmic wheat [6,64,65]. Our previous studies revealed that wheat class B MADS-box genes WPI1, WPI2 and WAP3 are downregulated at floral whorl three in alloplasmic wheat lines showing pistillody [6,14], whereas two class C MADSbox genes, WAG-1 and WAG-2, and TaDL, an ortholog of rice DROOPING LEAF (DL), are ectopically expressed in primordia of the pistil-like transformed stamens [15,16,23,66]. After floral organ identity is homeotically changed at whorl three, ovule formation is likely to automatically occur within the transformed stamens through expression of the class D MADS-box gene TaAGL2/WSTK [23]. During ectopic ovule development, expression of WBsis and WANT-1 is observed in integuments of the ovules [16,23]. WBLH2 was also strongly expressed in integuments not only of normal ovules in pistils but also of ectopic ovules in transformed stamens ( Figure 6). WBLH1/WBLH3/WBLH4 expression was observed in the basal boundary region of the ovary in both normal pistils and transformed stamens. These observations suggest that expression of the four WBLH genes is associated with development of transformed stamens in alloplasmic wheat lines with Ae. crassa cytoplasm.
Ectopic ovules in transformed stamens and ovules in pistils of (cr)-CSdt7BS are sterile due to abnormal development of the inner epidermis and integuments [6,23]. It was thought that aberrant expression of WBsis and a reduced transcript level of WANT-1 might at least partially result in the sterility of the ovules and ectopic ovules [16,23] (Murai et al. unpublished results). Such an obvious alteration of gene expression in the ovules and ectopic ovules was not observed in WBLH2. The WBLH2 expression pattern in the sterile pistils seemed to be identical to that in normal ovules of fertile pistils ( Figure 6D). Therefore, we have no evidence for WBLH2 being associated with the female sterility of the transformed stamens and pistils in (cr)-CSdt7BS. The regulatory pathway of WBLH2 expression in ovule development might be independent of those of WBsis and WANT-1. After WAG-1 and WAG-2 expression in the transformed stamens and sterile pistils, WSTK initiates ovule development [23]. WBLH2 expression occurs continuously in the developing ovules of the transformed stamens and sterile pistils, whereas WBsis is ectopically expressed in the marginal region of ovule primordia and WANT-1 expression level is reduced around ovule primordia [16,23] (Murai et al. unpublished results). After ovule initiation via WSTK, some of the ovule development-related genes such as WBsis and WANT-1 should be abnormally expressed, which in turn might be associated with the aberrant ovule development. In further studies, signal cascades from initiation of ovule primordia to WBsis/WANT-1 or WBLH2 expression should be elucidated to investigate how the Ae. crassa cytoplasm and Rfd1 affect female sterility in the pistillody-exhibiting alloplasmic wheat lines.