Common wheat is a hexaploid species with most of the genes present as triplicate homoeologs. Expression divergences of homoeologs are frequently observed in wheat, as well as in other polyploid plants. However, the mechanisms underlying this phenomenon are poorly understood.
Expansin genes play important roles in the regulation of cell size, as well as organ size. We found that all three TaEXPA1 homoeologs were silenced in seedling roots. In seedling leaves, TaEXPA1-A and TaEXPA1-D were expressed, but TaEXPA1-B was silenced. Further analysis revealed that silencing of TaEXPA1-B in leaves occurred after the formation of the hexaploid.
Chromatin immunoprecipitation assays revealed that the transcriptional silencing of three TaEXPA1 homoeologs in roots was correlated with an increased level of H3K9 dimethylation and decreased levels of H3K4 trimethylation and H3K9 acetylation. Reactivation of TaEXPA1-A and TaEXPA1-D expression in leaves was correlated with increased levels of H3K4 trimethylation and H3K9 acetylation, and decreased levels of H3K9 dimethylation in their promoters, respectively. Moreover, a higher level of cytosine methylation was detected in the promoter region of TaEXPA1-B, which may contribute to its silencing in leaves.
We demonstrated that epigenetic modifications contribute to the expression divergence of three TaEXPA1 homoeologs during wheat development.
Polyploidy refers to organisms containing more than two paired sets of chromosomes as a result of whole genome duplication (autopolyploid) or from the combination of two or more distinct but related genomes (allopolyploid). It has been demonstrated that c. 70% of flowering plants experienced polyploidization events during their evolution (Masterson, 1994; Wendel, 2000). Allopolyploidy is the most common form of polyploidy and can arise through two major events: one is hybridization of two different genomes into one nucleus, and the other is genome doubling occurring in interspecific hybrids (Grant, 1981; Thompson & Lumaret, 1992). When two or more different genomes are combined into a single cell, they must respond to the consequences of genome duplication, especially duplicate copies of genes with similar or redundant functions. There are three possible evolutionary fates for homoeologous genes in polyploids: retention of original or similar function, functional diversification and gene silencing (Wendel, 2000). Various mechanisms affect the fate of homoeologous genes, including changes in genome organization, altered regulatory interactions and epigenetic modifications (Adams & Wendel, 2005; Comai, 2005).
Wheat is a hexaploid species with the genome constitution of AABBDD originating from three diploid ancestral species: Triticum urartu, Aegilops speltoides and Aegilops tauschii (Feldman & Levy, 2005). Allopolyploidization leads to the generation of duplicated homoeologous genes (homoeologs). Therefore, most genes in hexaploid wheat are present as triplicate homoeologs. However, the presence of three homoeologs in wheat does not necessarily imply that three independent mRNAs are transcribed, and transcriptional divergence between homoeologous genes has been documented widely in hexaploid wheat (Bottley et al., 2006; Shitsukawa et al., 2007; Hovav et al., 2008; Chaudhary et al., 2009; Hu et al., 2011). Organ-specific regulation of homoeologous gene expression is also frequently observed (Bottley et al., 2006). The available data suggest that some changes may be attributed to DNA sequence alterations (Kashkush et al., 2002; Nie et al., 2008), whereas others are mediated by chromatin modifications and transcriptional regulation (Chen & Ni, 2006; Shitsukawa et al., 2007). For example, in hexaploid wheat, the protein encoded by WLHS1-A has no apparent function, whereas WLHS1-B is predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat (Shitsukawa et al., 2007). Recently, Zhang et al. (2011) reported a rare case in which the fate of a set of homoeologous genes was associated with plant morphology and domestication in hexaploid wheat. A recent mutation (V329I) in 5Aq was proposed to cause the ‘hyperfunctionalization’ of 5AQ; 5Bq may undergo pseudogenization, but remains transcriptionally active and influences the regulation of expression of the homoeologs; and 5Dq may have been subfunctionalized because of its association with many of the domestication traits.
Expansins are a family of closely related nonenzymatic proteins that induce cell wall extension and stress relaxation at acidic pH conditions in plants (McQueen-Mason et al., 1992). Based on their activities and sequence identities, expansins can be divided into two groups: α- and β-expansins (Shcherban et al., 1995; Lee et al., 2001; Cosgrove et al., 2002). It has been shown that expansins play important roles in plant development, including seed germination (Chen et al., 2001), root hair emergence (Zhang & Hasenstein, 2000; Cho & Cosgrove, 2002), leaf growth (Pien et al., 2001; Reidy et al., 2001), stem elongation (Cho & Kende, 1997), pollination (Cosgrove et al., 1997), floral opening (Gookin et al., 2003), meristem dynamics (Fleming et al., 1997), fruit softening (Brummell et al., 1999), as well as in adaptive responses to submergence, GA and wounding (Lee & Kende, 2001), and other developmental processes in which cell wall loosening occurs (Cosgrove, 2000).
In wheat, we have isolated previously 18 cDNAs encoding expansin proteins, and tissue-specific and developmentally restricted expression was detected for some expansin genes (Lin et al., 2005). Further analysis revealed that TaEXPA1 was highly expressed in young or vigorous tissues, but silenced in roots, and over-expression of TaEXPA1 in Arabidopsis increased the leaf size and root length (Z. Hu et al., unpublished). As a result of its important role in regulating cell and organ size, it is of great interest to reveal how the expression divergence of TaEXPA1 homoeologous genes is regulated in hexaploid wheat. In this study, we cloned three TaEXPA1 homoeologous genes and investigated their transcript levels in hexaploid wheat and three diploid progenitors. Moreover, we described the epigenetic regulation mechanisms for the tissue-specific expression divergence of three wheat expansin TaEXPA1 homoeologs.
Materials and Methods
The hexaploid wheat genotype Nongda 3338 (Triticum aestivum L.), diploid species BO5 (Triticum urartu, AA), SP6 (Aegilops speltoides, SS, possibly modified BB) and SQ523 (Ae. squarrosa, DD), and two sets of synthetic hexaploid wheat were used in this study. The first set included one 10th generation synthetic allohexaploid wheat SCAUP/SQ523 and its parental species durum wheat SCAUP (T. turgidum, AABB, 2n = 28) and goat grass SQ523 (Ae. tauschii, DD, 2n = 14), which were provided by the International Maize and Wheat Improvement Center (CIMMYT). The second set constituted the parental species tetraploid wheat DM4 (T. dicoccum, AABB, 2n = 28) and goat grass Y199 (Ae. tauschii, DD, 2n = 14), and their newly synthesized allohexaploid wheat (Nie et al., 2008).
Wheat seeds were germinated on moist filter papers and transplanted into pots to grow in the glasshouse under a 12-h photoperiod provided by cool white fluorescent and incandescent light (intensity ≥ 3000 lx). The plants were watered with 1/10-strength Hoagland's solution when necessary.
Cloning of wheat TaEXPA1 genes
The full-length cDNAs of TaEXPA1 homoeologous genes were obtained by PCR-directed cloning based on a wheat expressed sequence tag (EST) database (Lin et al., 2005). Full-length cDNAs were amplified with the following primer pair: TaEXPA1-L and TaEXPA1-R (Supporting Information Table S1). PCR products were subcloned and sequenced. The upstream and downstream regions of TaEXPA1 homoeologous genes were cloned using a SMART-rapid amplification of cDNA ends (SMART-RACE) cDNA amplification kit (Clontech, Mountain View, CA, USA) following the manufacturer's protocol. Gene-specific primers used for the first- and second-round PCRs were 5′-GSP1 and 5′-GSP2 (5′-RACE) and 3′-GSP1 and 3′-GSP2 (3′-RACE; Table S1), respectively. The promoter regions of TaEXPA1 homoeologous genes were cloned using the Universal GenomeWalker kit (BD Biosciences Clontech) according to the manufacturer's instructions employing the primers pro-GSP1, pro-GSP2-A and pro-GSP2-BD (Table S1).
RNA extraction and quantitative PCR analysis
Total RNA was isolated from tissues of wheat Nongda 3338 at various developmental stages, including dry seed embryos, shoots 1 d after germination (DAG), roots and fully expanded leaves at 10 DAG, stems in the jointing stage, flag leaves, immature ears, base of 1.5-cm-long portion at the first internode in the heading stage and seeds 6 d after pollination (DAP), using a Trizol RNA isolation protocol (Life Technologies, NY, USA). First-strand cDNA (Life Technologies) synthesis was performed using 2 μg of DNase-digested total RNA with oligo (dT) primer according to the protocol for reverse transcription-polymerase chain reaction (RT-PCR) first-strand synthesis (Promega, WI, USA).
Gene-specific primers for quantitative PCR (TaEXPA1-aL, TaEXPA1-AD-R and TaEXPA1-BD-R) were designed on the basis of the nucleotide polymorphisms in the cDNA sequences of the three TaEXPA1 homoeologous genes (Table S1), and the amplification efficiency of the primers was confirmed by homoeologous-specific genomic PCR in combination with denaturing high-performance liquid chromatography (dHPLC) analysis (Guo et al., 2003). For homoeologous-specific gene expression analysis, β-actin was amplified as an endogenous control, and three PCR replicates were performed for each cDNA sample. The RT-PCR products were then separated and quantified by the WAVE dHPLC system. Transcript abundance of EXPA1 homoeologs in the diploid species was amplified by semiquantitative PCR using the primer pair RT-EXPA-L and RT-EXPA-R (Table S1). Transcript levels of EXPA1-B in the tetraploid and the synthetic hexaploids were amplified by real-time PCR using the primer pair RT-EXPA-BL and RT-EXPA-BR (Table S1). The specificity of the primers was confirmed using plasmids containing full-length cDNA sequences of TaEXPA1 homoeologs, and β-actin was amplified as an endogenous control (Table S1).
Chromatin immunoprecipitation (ChIP) analysis
The ChIP assays were performed using a protocol modified from previously published methods (He et al., 2004; Tian et al., 2005). For each assay, fresh leaves and roots (c. 5 g) at 10 DAG were subjected to vacuum infiltration in a formaldehyde (1%) solution to crosslink the chromatin proteins to DNA. Chromatin was extracted and sonicated (Branson Sonifier, Danbury, CT, USA) at half maximal power for ten 10-s pulses with chilling on ice for 2 min after each pulse. The average size of the resultant DNA fragments produced was c. 0.2–1.5 kb. We used an aliquot of chromatin solution (1/10 of total volume) as input DNA to determine the DNA fragment sizes. The remaining chromatin solution was diluted 10-fold and divided into two aliquots: one was incubated using 10 μl of antibodies (anti-trimethyl-H3K4, anti-dimethyl-H3K9 or anti-acetyl-H3K9; Upstate Biotechnology, NY, USA) and the other was incubated without antibodies (mock). The immunoprecipitated DNA was used for semiquantitative PCR analysis. Homoeologous gene-specific primers were designed as follows: ChIP-EXPA1-AL and ChIP-EXPA1-AR; ChIP-EXPA1-BL and ChIP-EXPA1-BR; ChIP-EXPA1-DL and ChIP-EXPA1-DR (Table S1). The controls were the amplified DNA from the chromatin fractions before antibody incubation (Input) and from those that were precipitated without antibodies (Mock). β-actin (ChIP-β-actin-L and ChIP-β-actin-R) was amplified as an endogenous control for H3K4me3 and H3K9Ace. A retrotransposon gene (contig423638) was used as an endogenous control for H3K9me2 using the following primers: ChIP-Retro-L and ChIP-Retro-R (Table S1). Two independent experiments were performed for each assay with three biological replicates.
DNA isolation and bisulfite sequencing
Genomic DNAs were isolated from fully expanded leaves at 10 DAG using the DNeasy Plant Mini kit (Qiagen). The EZ DNA Methylation-Gold kit was used for bisulfite treatment of genomic DNA according to the manufacturer's instructions (ZYMO Research, CA, USA). The bisulfite-treated DNAs were then used for PCR amplification. The primer sets were designed using the web-based software MethPrimer (http://www.urogene.org/methprimer/index1.html; Table S1). PCR products were cloned into the pGEM-T plasmid vector (Promega) and sequenced by vector-specific primers. Sequencing data were analyzed using Kismeth software (Gruntman et al., 2008; http://katahdin.cshl.edu/homepage/kismeth/revpage.pl).
Identification of three homoeologous TaEXPA1 genes in wheat
Three homoeologs of TaEXPA1 were cloned and identified as 1624-, 1642- and 1663-bp gene fragments, respectively, by genomic PCR amplification based on our previously reported cDNA sequence of TaEXPA1 (GenBank accession number: AY485121; Lin et al., 2005). The preliminary chromosomal locations of these clones were determined by the second intron length polymorphisms in combination with Chinese Spring (CS) nulli-tetrasomics lines, and three TaEXPA1 genes were mapped on chromosomes 1A, 1B and 1D, respectively (Fig. 1). Further analysis using CS deletion lines in combination with the homoeolog-specific primer pair TaEXPA1-aL/TaEXPA1-BD-R showed that the PCR products of 1624 and 1663 bp were located on chromosomes 1BL and 1DL, respectively. Another fragment of 1642 bp was located on chromosome 1AL (Fig. S1). Therefore, we concluded that these three genes were homoeologous, and designated them as TaEXPA1-A, TaEXPA1-B and TaEXPA1-D, respectively.
To obtain the full-length cDNA corresponding to the three homoeologous genes, 5′ and 3′ RACE was performed. The 5′ and 3′ sequences obtained were aligned with the sequences of TaEXPA1 homoeologous cDNAs, and three full-length cDNAs of 1251, 1218 and 1189 bp were obtained, respectively. A sequence comparison between cDNA and genomic DNA revealed that TaEXPA1 has three exons and two introns (Fig. S2). Further analysis revealed that the coding sequences (CDS) of the three TaEXPA1 homoeologs were highly conserved, and can only be distinguished from one another by virtue of single nucleotide polymorphisms (SNPs) and a CTT indel between TaEXPA1-A and TaEXPA1-D. Deduced amino acid sequence alignments also showed that there were no significant differences among the three TaEXPA1 homoeologs (Fig. S3). In contrast with the CDS, differences in molecular size were found at two introns and GAA microsatellite repeats in the 3′-untranslated region (3′-UTR; Fig. S2).
To gain further insight into the nucleotide sequence divergence of TaEXPA1 homoeologs, the genomic DNA and cDNA of leaves from three ancestral diploid species, BO5 (Triticum urartu, AA), SP6 (Aegilops speltoides, SS) and SQ523 (Ae. squarrosa, DD), were obtained and named as EXPA1-AA, EXPA1-SS and EXPA1-DD, respectively. Sequence alignments indicated that TaEXPA1-A, TaEXPA1-B and TaEXPA1-D had similar genomic structures to EXPA1-AA, EXPA1-SS and EXPA1-DD from ancestral species, respectively (Fig. S2).
Expression divergence of the three TaEXPA1 homoeologous genes
To examine the expression profiles of TaEXPA1 homoeologs in different organs at different developmental stages, gene-specific PCR primers were designed and the amplification efficiency of the primers was confirmed by homoeolog-specific genomic PCR in combination with dHPLC analysis. As shown in Fig. 2(a), the primer pairs TaEXPA1-aL/TaEXPA1-AD-R and TaEXPA1-aL/TaEXPA1-BD-R can be used for subsequent expression analysis (Fig. 2a). The results indicated that all three TaEXPA1 homoeologous genes were expressed in dry seed embryos, shoots of 1-DAG seedlings, jointing stage stems, heading stage flag leaves and heading stage immature ears. However, the expression levels of the three TaEXPA1 homoeologs were significantly different in different tissues and different development stages. For example, TaEXPA1-A and TaEXPA1-D were expressed at higher levels than TaEXPA1-B in shoots of 1-DAG seedlings and heading stage flag leaves and immature ears (Fig. 2b), whereas TaEXPA1-A was not expressed in the first internode at the heading stage or in seeds at 6 DAP, but TaEXPA1-B and TaEXPA1-D were highly expressed in the same tissues (Fig. 2b). Most notably, all three TaEXPA1 homoeologs were silenced in seedling roots at 10 DAG. In the leaves of the same stage, TaEXPA1-A and TaEXPA1-D were expressed, but TaEXPA1-B was silenced (Fig. 2b). Taken together, we concluded that the expression of the three TaEXPA1 homoeologs varied dynamically at different stages and organs during wheat development.
In addition, we investigated the transcript levels of TaEXPA1 genes in three diploid progenitors of hexaploid wheat to reveal whether the differential transcript levels of the three homoeologs in hexaploid wheat reflect the respective transcription levels of those in diploid species. The transcript profiles in seedling roots of the three diploids resembled those in the three genomes of hexaploid wheat, suggesting that silencing of TaEXPA1 in seedling roots is evolutionarily conserved. Interestingly, although TaEXPA1-B was silenced in seedling leaves in hexaploid wheat, TaEXPA1 was transcribed in all diploid species. Studies of gene expression in natural and synthetic polyploids have shown that some genes are silenced after polyploidization (Bottley et al., 2006; Nie et al., 2008). Thus, we further investigated the expression patterns of TaEXPA1-B in seedling leaves of two synthetic hexaploid wheats (AABBDD) and their tetraploid parents (AABB). The results revealed that TaEXPA1-B was highly expressed in the two tetraploids (DM4 and SCAUP), but was down-regulated in the synthetic hexaploids (Fig. 2d), indicating that silencing of TaEXPA1-B in leaves occurred after the formation of the hexaploid. It should be noted that the expression of TaEXPA1-B in the synthetic hexaploids was detected even though it is completely silenced in the natural hexaploids. One possible explanation is that the expression of TaEXPA1-B decreased after polyploidization by initial epigenetic modifications, and was then silenced sometime during the evolution of the hexaploid wheat.
Expression divergence of TaEXPA1 homoeologs is mediated by histone methylation and acetylation
In order to understand the mechanisms of transcriptional silencing of TaEXPA1 in roots, promoter sequences of three TaEXPA1 homoeologs were cloned and aligned. The results demonstrated that there were nucleotide polymorphisms present, including eight indels residing upstream in the promoter region, but neither crucial indels nor specific mutations of known cis-elements were detected using the PLANTCARE website (Fig. 3). Recent work has shown that epigenetic changes may play a crucial role in maintaining homolog expression patterns in allopolyploid plants (Chen, 2007; Gaeta et al., 2007; Shitsukawa et al., 2007; Chen et al., 2011). We determined the levels of H3K4 trimethylation, H3K9 dimethylation and H3K9 acetylation using ChIP assays and semiquantitative PCR amplification with gene-specific primers designed for TaEXPA1-A, TaEXPA1-B and TaEXPA1-D. We found that H3K9me2 levels were significantly higher in the promoter sequences of the three TaEXPA1 homoeologs in roots when compared with leaves; however, the levels of H3K4me3 and H3K9ac were significantly lower in the promoter sequences of the three TaEXPA1 homoeologs in roots than in leaves (Fig. 4). We also found that the H3K4me3 and H3K9ac levels were higher in the promoter sequences of TaEXPA1-A and TaEXPA1-D in leaves than in roots. By contrast, the level of H3K9me2 was lower in the promoter sequences of TaEXPA1-A and TaEXPA1-D in leaves than in roots (Fig. 4). As no differences were found in the known cis-elements in the promoter sequences for the three homoeologous genes, we concluded that the transcriptional silencing in roots was associated with increased levels of H3K9 dimethylation and decreased levels of H3K4 trimethylation and H3K9 acetylation in the promoter. The reactivation of TaEXPA1-A and TaEXPA1-D expression in leaves was correlated with increased H3K4 trimethylation and H3K9 acetylation, and decreased levels of H3K9 dimethylation, in the promoter, respectively. These data suggested that the tissue-specific expression of the three TaEXPA1 homoeologs was mediated by histone methylation at H3K4 and H3K9 and acetylation at H3K9 sites.
TaEXPA1-B silencing in leaves is related to DNA methylation
Sequence analysis indicated that the promoter regions of the three TaEXPA1 homoeologous genes had > 60% GC content, which is a criterion for CpG islands. Therefore, the DNA methylation status in the promoter regions of the three TaEXPA1 homoeologs was examined using bisulfite PCR analysis of CG/CHG/CHH sites. In this study, we found that the methylation status of CG/CHG sites in the promoter region of TaEXPA1-B was higher when compared with its homoeologs TaEXPA1-A and TaEXPA1-D (Fig. 5a–c), which could contribute to its silencing in leaves. In addition, we examined methylation levels of the EXPA1-B promoter in tetraploids (Triticum dicoccum, AABB) and synthetic hexaploids (AABBDD) in leaves of 10-DAG seedlings. This analysis indicated that the methylation status of CHG/CHH sites in the promoter region of EXPA1-B was higher in synthetic hexaploids relative to tetraploids, which seems to be associated with down-regulation of the gene in synthetic hexaploids (Fig. 5d,e).
Expression divergence of the three TaEXPA1 homoeologs
In this study, we identified three TaEXPA1 sequences (TaEXPA1-A, TaEXPA1-B and TaEXPA1-D), and located these genes on wheat chromosomes 1A, 1B and 1D, respectively. Numerous studies have shown that polyploidization is often accompanied by changes in genomic structure and gene expression, including loss of function, gene silencing and homoeologous gene expression diversification (Wendel, 2000; Bottley et al., 2006; Hovav et al., 2008; Chaudhary et al., 2009). For example, a comparison of three WLHS1 homoeologous gene sequences in wheat indicated that WLHS1-A has a 3479-bp insertion, leading to the loss of gene function (Shitsukawa et al., 2007). A systematic study has revealed that homoeologous sequences of benzoxazinone (Bx) biosynthetic genes (TaBx1–TaBx5) are highly conserved in the coding nucleotide sequences and in the exon/intron structures, but are transcribed differentially, with the homoeologs from the B genome generally contributing the most to Bx biosynthesis in hexaploid wheat (Nomura et al., 2005). Recently, Zhang et al. (2011) reported that the evolution of the Q/q loci in polyploid wheat resulted in the hyperfunctionalization of 5AQ, pseudogenization of 5Bq and subfunctionalization of 5Dq, all contributing to domestication traits. Most notably, expression analysis indicated that the homoeoalleles of the Q/q gene were co-regulated in a complex manner, which may be caused by dosage balance or complementary effects among the Q/q homoeoalleles. In this study, we found that the three TaEXPA1 homoeologs had a similar genomic structure in the coding region with no apparent insertions or deletions detected. However, significant differences in the expression patterns of the three TaEXPA1 homoeologs were observed in wheat (Fig. 2b). For example, TaEXPA1-A and TaEXPA1-D were expressed in seedling leaves at 10 DAG, but TaEXPA1-B was silenced. Further analysis indicated that silencing of TaEXPA1-B in leaves occurred after the formation of the hexaploid. The expression of duplicated genes resulting from polyploidization (termed homoeologs) can be partitioned between the duplicates, so that only one copy is expressed and functions only in some organs, whereas the other copy is expressed only in other organs (Adams et al., 2003). Therefore, our study provided an example of partitioning of homoeolog gene expression in wheat, which may lead to gene retention during subsequent evolution (Lynch & Force, 2000; Adams et al., 2003; Liu & Adams, 2007).
Tissue-specific expression of TaEXPA1 homoeologs is caused by epigenetic regulation
Studies have shown that homoeolog silencing is organ or tissue specific in polyploids, with one homoeolog silenced in leaf tissue, but expressed in roots, or vice versa (Adams et al., 2003; Bottley et al., 2006). However, the mechanisms of expression divergence for homoeologs are still poorly understood. Histone modifications have been demonstrated to mediate the epigenetic regulation of gene expression, growth and development in plants and animals (Berger, 2007; Li et al., 2007; Zhang, 2008). Acetylation of histone H3 and trimethylation of H3 lysine 4 are known as euchromatic marks and are often associated with active transcriptions, whereas methylation of H3K9 and H3K27 are known as heterochromatic marks and are related to gene repression (Jenuwein & Allis, 2001; Li et al., 2007). Our analysis indicated that TaEXPA1-A and TaEXPA1-D were highly expressed in leaves of 10-DAG seedlings, but the transcript level of TaEXPA1-B was clearly lower than that of its counterparts. All three TaEXPA1 homoeologs were silenced in roots during the same period. Using ChIP analysis, we found that transcriptional silencing of the three TaEXPA1 homoeologs in roots was associated with increased levels of H3K9me2 and decreased levels of H3K4me3 and H3K9ac in the promoter regions. Reactivation of TaEXPA1-A and TaEXPA1-D expression in leaves was correlated with increased H3K4me3 and H3K9ac levels, and decreased levels of H3K9me2, in the promoters, respectively. These results indicated that epigenetic regulation contributed to the tissue-specific expression of the three TaEXPA1 homoeologs. It should be noted that the observed alterations in histone modifications only explain differences in expression between roots and leaves, but not the silencing of TaEXPA1-B in leaves.
Evolutionary origin of TaEXPA1-B silencing in leaves
Expression analysis indicated that TaEXPA1-A and TaEXPA1-D were highly expressed, but TaEXPA1-B was silenced, in seedling leaves at 10 DAG. Silencing of EXPA1-B was not observed in the diploid species SP6, the putative B genome donor of the hexaploid wheat. Furthermore, EXPA1-B was expressed abundantly in two tetraploids (DM4 and SCAUP), but was down-regulated in the synthetic hexaploids (DM4/Y199 and SCAUP/SQ523; Fig. 2d). These results suggested that silencing of TaEXPA1-B occurred subsequently after the formation of the hexaploids.
Most angiosperms probably experienced a polyploidy event(s) at some point during their evolutionary history (Masterson, 1994). One direct and observable consequence of polyploidy is transcriptional gene silencing, and this phenomenon has been widely confirmed in allotetraploid Arabidopsis (Comai et al., 2000), cotton (Adams et al., 2003) and wheat (Kashkush et al., 2002; Shitsukawa et al., 2007). These reports indicated that the silencing was achieved by epigenetic rather than genetic modifications (Shitsukawa et al., 2007). Using bisulfite genomic sequencing analysis, we examined the methylation levels of the CG, CHG and CHH islands of the three TaEXPA1 homoeologs. This analysis indicated that gene-specific CG and CHG hypermethylation was present in the promoter of TaEXPA1-B, which could be associated with the silencing of this gene (Fig. 5a–c). We also found that the methylation status of CHG/CHH sites in the promoter region of EXPA1-B was lower in the tetraploid (Triticum dicoccum, AABB) than in the synthetic hexaploid (AABBDD; Fig. 5d,e), suggesting that the epigenetic regulation of TaEXPA1-B occurred after the formation of the hexaploid. This result was also consistent with a previous study showing that hexaploid wheat formation is accompanied by a general increase in DNA methylation when compared with tetraploid wheat (Zhao et al., 2011).
Roles of TaEXPA1 genes in plant development
Expansin genes have been isolated from a variety of plant species and are a large multigene family (Cosgrove, 1998; Wu et al., 2001; Li et al., 2002). Different members of expansin are expressed in various parts of plants at different developmental stages. For example, LeEXP4 has been shown to be expressed specifically in the endosperm cap tissue enclosing the radicle tip (Chen & Bradford, 2000). However, LeEXP8 and LeEXP10 are expressed in the embryo. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest that they play a role in the initial elongation stage of the radicle and seedling growth (Chen et al., 2001). The complexity of expansin gene expression is reflected in the similarly diverse expression patterns found in wheat, in which three TaEXPA1 homoeologs were differentially expressed in different tissues and developmental stages. The distinct spatial and temporal expression patterns of the three TaEXPA1 homoeologs suggest that TaEXPA1 may play multiple roles in wheat development.
Previous studies have suggested that expansins function in the control of plant cell growth (Fleming et al., 1997; Cho & Cosgrove, 2000; Li et al., 2003). GmEXP1 is expressed mainly in regions undergoing cell division and elongation in the primary and secondary roots, suggesting a role in root elongation in soybean (Lee et al., 2003).
In addition, in silico analysis has indicated that TaEXPA1 is orthologous to AtEXP10 in Arabidopsis. AtEXP10 is expressed predominantly in young growing petioles and leaf blades. A previous study has demonstrated that leaf size is substantially reduced in antisense lines with suppressed AtEXP10 expression (Cho & Cosgrove, 2000). Overexpression of AtEXP10 produces larger rosette leaves and the plants mature earlier when compared with wild-type plants. Interestingly, overexpression of the three TaEXPA1 homoeologs in Arabidopsis increased leaf size during the seedling and adult stages, suggesting that TaEXPA1 genes may play an important biological role in the control of leaf growth in wheat (Z. Hu et al., unpublished). However, the results of the ecotopic overexpression of TaEXPA1 genes in Arabidopsis may not reflect their functions during wheat development. Therefore, the roles of TaEXPA1 genes in wheat leaf growth need to be elucidated further.
We gratefully acknowledge Dr Xueyong Zhang (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China) for providing the seeds of diploid species BO5 and SP6. This work was financially supported by the Major Program of the National Natural Science Foundation of China (31290210), National Basic Research Program of China (973 Program) (2012CB910900) and National Science Foundation for Distinguished Young Scholars (30925023).