The study of two barley type I-like MADS-box genes as potential targets of epigenetic regulation during seed development

Abstract

Background: MADS-box genes constitute a large family of transcription factors functioning as key regulators of many processes during plant vegetative and reproductive development. Type II MADS-box genes have been intensively investigated and are mostly involved in vegetative and flowering development. A growing number of studies of Type I MADS-box genes in Arabidopsis, have assigned crucial roles for these genes in gamete and seed development and have demonstrated that a number of Type I MADS-box genes are epigenetically regulated by DNA methylation and histone modifications. However, reports on agronomically important cereals such as barley and wheat are scarce.

Results: Here we report the identification and characterization of two Type I-like MADS-box genes, from barley (Hordeum vulgare), a monocot cereal crop of high agronomic importance. Protein sequence and phylogenetic analysis showed that the putative proteins are related to Type I MADS-box proteins, and classified them in a distinct cereal clade. Significant differences in gene expression among seed developmental stages and between barley cultivars with varying seed size were revealed for both genes. One of these genes was shown to be induced by the seed development- and stress-related hormones ABA and JA whereas in situ hybridizations localized the other gene to specific endosperm sub-compartments. The genomic organization of the latter has high conservation with the cereal Type I-like MADS-box homologues and the chromosomal position of both genes is close to markers associated with seed quality traits. DNA methylation differences are present in the upstream and downstream regulatory regions of the barley Type I-like MADS-box genes in two different developmental stages and in response to ABA treatment which may be associated with gene expression differences.

Conclusions: Two barley MADS-box genes were studied that are related to Type I MADS-box genes. Differential expression in different seed developmental stages as well as in barley cultivars with different seed size was evidenced for both genes. The two barley Type I MADS-box genes were found to be induced by ABA and JA. DNA methylation differences in different seed developmental stages and after exogenous application of ABA is suggestive of epigenetic regulation of gene expression. The study of barley Type I-like MADS-box genes extends our investigations of gene regulation during endosperm and seed development in a monocot crop like barley.

Figure 1

Amino acid sequence comparisons between Type I MADS-box proteins and Type II MADS-box proteins from different plants. The name of each sequence consists of its Uniprot ID or GenBank accession number, followed by the species abbreviation according to Uniprot. The K-box domains, K1, K2 and K3 of the Type II MADS-box proteins are shown in boxes and indicated with red bars. The MADS-box domain is also indicated with a red bar. No K-box is present in the Type-I proteins. Identical amino acids are shown in dark blue and similar amino acids in light blue.

Figure 2

Amino acid sequence alignments between barley HvOS1, HvOS2, and Type I proteins from different plants. The name of each sequence consists of its GenBank accession number, also shown in Table 1. Identical amino acids are shown in dark blue and similar amino acids in light blue. The MADS-box domain is indicated with a red bar.

Figure 3

Phylogenetic tree of MADS-box proteins from different plants. Phylogenetic tree showing the different clades of MADS-box Type I family proteins. The Type II MADS-box SEPALATA, AGAMOUS and SOC1 families are shown as a condensed branch. The sequences used and their accession numbers are shown in Table 1. Barley HvOS1 and HvOS2 members are in bold. Numbers indicate bootstrap values (1000 = 100%).

Figure 4

Expression analysis of barley Type I-like HvMADS-box genes. A) Qualitative RT-PCR expression analysis of HvOS1 and HvOS2 barley Type I-like HvMADS-box genes. R, roots; AM, apical meristem; YS, young shoots; L, Leaves; IF, immature flower; HvActin, was used as the internal control. B) Quantitative real-time RT-PCR analysis of Type I-like HvMADS-box genes in different seed developmental stages and in two barley cultivars. Expression values were normalized to those of HvActin. The relative expression ratio of each sample is compared to the control group which was C5 (Caresse immature flowers). C, cultivar Caresse, (white bars); IP, cultivar Ippolytos (grey bars). 5, Immature flowers; 6, Seed 1–3 DAF; 7, Seed 3–5 DAF; 8, Seed 5–10 DAF; 9, Seed 10–15 DAF; 10, Seed 15–20 DAF. Data represent mean values from two independent experiments with standard deviations. Values significantly different (P < 0.05) from the control group (C5) are marked with an asterisk. C) Spatial expression of HvOS2 in barley seeds. In situ localization of HvOS2 in barley seeds by mRNA in situ hybridization analysis. Left, transverse section of developing seeds at 10 DAF hybridized with the antisense probe. Right, transverse section of developing seeds at 10 DAF hybridized with the sense probe, as negative control. a, aleurone; e, endosperm; p, pericarp.

Figure 5

Expression analysis of HvOS1, HvOS2 after treatment of seedlings with ABA and JA. A). Quantitative real-time PCR analysis of HvOS1 and HvOS2 genes at 6 and 24 h after treatment of Caresse seedlings with 100 μM ABA and JA respectively. Grey and black bars, ABA and JA hormone-treated plants, respectively; white bars, no hormone treated plants (mocked with H₂O/Tween for 6 h and 24 h, respectively). B) Quantitative real-time PCR analysis of the barley genes HVA22 (known to be induced by ABA) and HvADC2 (known to be induced by JA) at 6 and 24 h after treatment of Caresse seedlings with 100 μM ABA and 100 μM JA, respectively, used as positive controls. Expression values were normalized to those of HvActin. Data represent mean values from two independent experiments with standard deviations. Relative expression ratio of each sample was compared to the control group which was untreated plants, 0 h, and was assigned the value of 1. Values significantly different (P < 0.05) from the control group (untreated 0 h) are marked with an asterisk.

Figure 6

Genomic organization of HvOS2 in relation to its cereal homologues. A) Schematic view of gene bodies (exons plus introns) of five cereal MADS-I-like genes. Exons are depicted with orange boxes and introns with blue lines. Numbers on start and end of contigs refer to the length of particular genomic region. Length (bp) of first large introns is given under each intron. Regions within the first intron where retrotransposons were found are highlighted in red. B) Sequence similarity of the first introns of maize ZmB4FML1 and brachypodium Bradi2g59120 with retrotransposons, visualized in Circoletto (http://tools.bat.ina.certh.gr/circoletto/) after blast analysis with MASiVE (http://tools.bat.ina.certh.gr/masive/) and LTRphyler (http://tools.bat.ina.certh.gr/ltrphyler/). Upper circle: Left, full length maize retrotransposon Copia. PZMAY_CS_U68408_OPIE detected within the first intron of maize ZmB4FML1 and depicted in red; Right, the first intron of maize ZmB4FML1. The region of retrotransposon homology is shown in red. Bottom circle: Left, the brachypodium retrotransposon, Gypsy PBDIS_GX_ADDN0100106. Right, the first intron of brachypodium Bradi2g59120. Regions of homology are shown in red. Coding regions of retrotransposons integrase and reverse transcriptase are in purple and yellow-green, respectively. The 5’and 3’ LTR regions of retrotransposons are shown with bold black lines. Dark grey and light grey interconnecting ribbons depict regions of high and low similarity, respectively.

Figure 7

Schematic diagram of 5’ upstream and 3’ downstream regions of HvOS1 and HvOS2. Regions 5’ upstream from the ATG translation initiation site, coding regions, and 3’downstream regions are depicted with thick bold black lines. Translation initiation codons, ATG, and stop codons TAG and TGA, respectively, are indicated. Cis acting elements are depicted in colour-coded boxes. Predicted CpG islands and regions used in DNA methylation assays are also depicted with thin black lines.

Figure 8

DNA methylation assays in seed stages. Genomic DNA was digested with Mcr BC and PCR-amplification followed. (−), no McrBC; (+), digestion with McrBC; IF, immature flower; 1–3 DAF, 1–3 days after fertilization. The regions analyzed are depicted schematically above the gel images for each gene. White boxes represent exons.

Figure 9

DNA methylation assays after ABA treatment. Genomic DNA from untreated and ABA-treated seedlings, at 24 h after treatment, was digested with McrBC and PCR-amplification followed. (−), no McrBC; (+), digestion with McrBC; The regions analyzed are depicted schematically above the gel images.