Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae

Abstract

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play an important role in post-transcriptional gene regulation in plants and animals by targeting messenger RNAs (mRNAs) for cleavage or repressing translation of specific mRNAs. The first miRNA identified in plants, miRNA156 (miR156), targets the SQUAMOSA promoter-binding protein-like (SPL) transcription factors, which play critical roles in plant phase transition, flower and plant architecture, and fruit development. We identified multiple copies of MIR156 and SPL in the rice, Brachypodium, sorghum, maize, and foxtail millet genomes. Sequence and chromosomal synteny analysis showed that both MIR156s and SPLs are conserved across species in the grass family. Analysis of expression data of the SPLs in eleven juvenile and adult rice tissues revealed that four non-miR156-targeted genes were highly expressed and three miR156-targeted genes were only slightly expressed in all tissues/developmental stages. The remaining SPLs were highly expressed in the juvenile stage, but their expression was lower in the adult stage. It has been proposed that under strong selective pressure, non-miR156-targeted mRNA may be able to re-structure to form a miRNA-responsive element. In our analysis, some non-miR156-targeted SPLs (SPL5/8/10) had gene structure and gene expression patterns similar to those of miR156-targeted genes, suggesting that they could diversify into miR156-targeted genes. DNA methylation profiles of SPLs and MIR156s in different rice tissues showed diverse methylation patterns, and hypomethylation of non-CG sites was observed in rice endosperm. Our findings suggested that MIR156s and SPLs had different origination and evolutionary mechanisms: the SPLs appear to have resulted from vertical evolution, whereas MIR156s appear to have resulted from strong evolutionary selection on mature sequences.

Keywords: DNA methylation; Gene expression; Grass genome; MicroRNA156 (miR156); SQUAMOSA promoter-binding protein-like (SPL) gene.

Fig. 1. Sequence alignment of MIR156 collinear regions in maize, sorghum, foxtail millet, rice, and Brachypodium. osa-MIR156s were used as the reference sequences to search for orthologous genes in other grass species. The MIR156s are shown in colored boxes according to their annotation names in miRBase, whereas other conserved syntenic genes are in black. The numbers represent the relative positions of regions on the chromosomes (Chrs).

Fig. 2. Phylogenetic analysis of MIR156s from members of the grass family. The phylogenetic tree was constructed with 52 MIR156 stem-loop sequences from the grass family. G1, MIR156a/b/c; G2, MIR156f/g; G3, MIR156d; G4, MIR156k; G5, MIR156e/h/i. The tree shown had the highest log likelihood (-891.7215) and bootstrap values of >50%.

Fig. 3. Alignments of miR156 mature sequences and putative targets in maize. (a) Comparison of miR156 mature sequences from grass species and Arabidopsis. Most mature sequences start with a U residue, which is one of the defining characteristics of miRNAs in plants (Wu et al., 2009). 1: bdi-miR156b‒i, osa-miR156a‒j, sbi-miR156a‒c, sbi-miR156f‒i, zea-miR156a‒i, zea-miR156l, ath-miR156a‒f; 2: bdi-miR156j, osa-miR156k, sbi-miR156d, zea-miR156j; 3: osa-miR156l; 4: sbi-miR156e, zea-miR156k; 5: ath-miR156g; 6: ath-miR156h; 7: ath-miR156i; 8: ath-miR156j. (b) Predicted putative targets of miR156 in maize. Black and gray shading indicate 100% and 80% conserved nucleotides, respectively.

Fig. 4. Phylogenetic analysis of SQUAMOSA promoter-binding protein-like (SPL) genes from grass, Arabidopsis, and moss. (a) A phylogenetic tree constructed from 102 SPL sequences from grass, 17 from Arabidopsis, 14 from moss, and 1 from green algae (as an out-group). The genes fall into eight lineages (G1‒G8), some of which are targeted by miRNA156 (G1, G5-G7) and some of which are not (G2‒G4, G8). (b) Gene structure of the SPLs. Gray filled boxes, SBP domains; White boxes, other exon regions; Lines, introns.

Fig. 5. WebLogo representation of the SQUAMOSA promoter-binding protein (SBP) domain sequences within SQUAMOSA promoter-binding protein-like(SPL) proteins of several grass species. The overall height of each stack within the 80-amino-acid SBP domain sequence reflects the conservation at the designated position, whereas the height of each single-letter amino acid symbol within each stack indicates the frequency of that amino acid. Two Zn²⁺-binding sites, Cys-Cys-His-Cys and Cys-Cys-Cys-His, are marked by the black lines above the sequence, and the nuclear localization signal (NLS) is marked by the gray lines below the sequence.

Fig. 6. Heatmap representation of SQUAMOSA promoter-binding protein-like(SPL)expression in different rice tissues and at developmental stages. The rice life cycle is divided into three phases: vegetative (1‒3), reproductive (4‒6), and gametophytic phase and seed maturation (9‒11). Expression data for OsSPLs in 11 tissues/developmental stages were obtained from the Gene Expression Omnibus (GEO) database. Relative gene expression levels from low (green) to high (red) are shown. The miR156-targeted genes are indicated by asterisks with red bold font, and non-targeted genes with normal font. 1. shoots; 2. leaves-20 d; 3. seedling four-leaf stage; 4. pistil; 5. pre-emergence inflorescence; 6. post-emergence inflorescence; 7. anther; 8. seed-5 DAP; 9. seed-10 DAP; 10. embryo-25 DAP; 11. endosperm-25 DAP. DAP: day after pollination.

Fig. 7. Methylation analysis of OsSPLs and osa-MIR156sin four rice tissues (embryo, endosperm, root, and shoot). (a, b) OsSPL11 (a) is an example of an SQUAMOSA promoter-binding protein-like(SPL) that is highly methylated within its putative promoter region and sparsely methylated within its gene body and 3'-untranslated region (3'-UTR), whereas OsSPL6 (b) is an example of biased methylation within the gene body and hypomethylation in the promoter and 3'-UTR; (c) osa-MIR156g exemplifies the methylation bias upstream of the hairpin (HP) structure and the absence of methylcytosine in the stem-loop sequences that are observed in osa-miR156 genes. The boxed regions of the methylation graphs indicate which regions are presented at higher resolution in the accompanying graphs. Note that the full-length sequence shows all three methylation sites. The gene structures are shown at the bottom of each subpanel. Both the SPLs and MIR156s are less frequently methylated in endosperm than in other tissues.

Fig. 8. Model for the evolutionary diversification of SQUAMOSA promoter-binding protein-like (SPL) gene structure. In this model, the original SPL was not targeted by miR156 and was made up of multiple exons (>10 exons). This gene then diversified into non-miR156-targeted (>10 exons) and miR156-targeted (2‒4 exons) genes containing microRNA responsive elements (MREs). Under strong environmental pressure, however, alternate splicing may have allowed non-miR156-targeted SPLs to reduce their number of exons, which could potentially have enabled them to form MREs, thus transforming them into miR156-targeted genes. The gray boxes indicate the SQUAMOSA promoter-binding protein (SBP) domains, and all exons and introns are shown in boxes and lines.