Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice

Abstract

Transcription factors play essential roles in the developmental processes of plants. Many such factors are regulated by microRNAs (miRNAs). SQUAMOSA (SQUA) promoter-binding-like (SPL) genes encode plant-specific transcription factors, some of which contain complementary sequences of miRNA156. In this study, 19 rice (Oryza sativa) SPL (OsSPL) genes and 12 rice miRNA156 (OsmiR156) precursors were identified in the rice genome. Sequence and experimental analysis suggested that 11 OsSPL genes were putative targets of OsmiR156. Plant SPL proteins were classified into six subgroups based on the phylogenetic analysis of SQUA promoter-binding protein domain. Diverse exon-intron structures and distinct organizations of putative motifs beyond the SQUA promoter-binding protein domains were identified in the OsSPL gene family. Transcript level analysis of OsSPL genes in various rice tissues and organs revealed different tempospatial expression patterns. More than half of the OsSPL genes including most OsmiR156-targeted genes are predominantly expressed in the young panicles, whereas OsmiR156 genes are predominantly expressed in the young shoots and leaves of rice. Overexpression of two OsmiR156 genes (OsmiR156b and OsmiR156h) in rice resulted in severe dwarfism, strongly reduced panicle size, and delayed flowering, suggesting that OsmiR156 and OsSPL target genes are involved in various developmental processes, especially the flower development of rice. Different patterns of transcript changes (decreased or unchanged) of different target genes in same tissue and of same target gene in different tissues detected in the OsmiR156-overexpressing plants suggested diverse interactions between OsmiR156 and OsSPL target genes in a tissue-specific manner.

Figure 1.

Sequence analysis of OsmiR156 genes. A, Sequence alignment of OsmiR156 mature sequences with complementary sequences of OsSPL genes. The conserved amino acid sequence encoded by the target sequences is shown at the bottom. The dots between miR156 and targeted OsSPL sequences indicate mismatches. The complementary sequences of OsmiR156 in OsSPL4 and OsSPL13 (indicated by an asterisk) are not in the coding regions. B, Distribution of OsSPL and OsmiR156 genes in rice genome. Locations of closely linked genes are magnified. OsmiR156b and OsmiR156c are mapped to the same transcript (corresponding to the cDNA AK110791). OsmiR156j and OsmiR156h are also mapped to same transcript (corresponding to the cDNA AK103769). OsSPL4 and OsSPL5 are closely linked with OsmiR156d, and OsSPL17 and OsSPL18 are closely linked with OsmiR156k. The putative target genes of OsmiR156 are underlined.

Figure 2.

Unrooted tree of SPL family based on the protein sequences of SBP domains. The numbers at branching sites indicated the posterior probability values for nodal support. The SPL genes from Arabidopsis and rice that contained complementary sequence of miR156 were underlined. The names of Arabidopsis SPL genes were downloaded from the Arabidopsis Functional Genomics Network (http://web.uni-frankfurt.de/fb15/botanik/mcb/AFGN/Huijser.htm); other plant SPL genes are from SwissProt. The SBP domain sequences of all these genes and the accession numbers of genes from plants other than rice are listed in Supplemental Table I. The accession numbers of SPL genes of rice are listed in Table I. Am, A. majus; At, Arabidopsis; Zm, maize; Bp, Betu psy; Os, rice.

Figure 3.

Organization of putative motifs in OsSPLs. A, Sequence LOGO view of the consensus SBP domain sequences based on the 48 plant SPLs in Figure 2 (pHMM logo view of each SPL subgroup is shown in Supplemental Fig. 1). The height of the letter (amino acid) at each position represents the degree of conservation. The rectangles at the top of the plot indicate the motifs of the SBP domain. B, Putative motifs in OsSPLs identified by MEME. Numbered color boxes represent different putative motifs (annotations are listed in Table II). The expected values calculated by MEME are shown after gene names. S1 to S6 indicates the phylogenetic subgroups from Figure 2. The putative target genes of OsmiR156 are underlined.

Figure 4.

Tissue-specific expression patterns of OsSPL genes and OsmiR156. A, Semiquantitative RT-PCR of OsSPLs and OsmiR156 precursors in 13 different tissues or organs of rice. The rice Actin1 gene was used as the internal control. The miR156-targeted OsSPL genes were underlined and two pairs of primers (Supplemental Table I) were used for each gene, one located downstream of the miR156 complementary site and the other (labeled with an asterisk) amplifying the region covering the targeting site. PCR was failed for OsSPL17 with the target site-spanning primers. B, Real-time quantitative PCR analysis of four OsSPL genes. The relative expression levels were calibrated based on sample 6 (0.5–1 cm panicle). C, PAGE RNA gel-blot analysis of mature miR156 using an end-labeled complementary sequence (5′-GTGCTCACTCTCTTCTGTCA-3′). The blot was stripped and rehybridized with oligonucleotide probe complementary to U6 RNA as a loading control. Amount of RNA loaded in each lane was indicated by ethidium bromide staining. Tissues or organs: 1, Root at tillering stage; 2, stems at tillering stage; 3, leaf sheath at 4-leaf stage; 4, leaf lamina at 4-leaf stage; 5, young panicle shorter than 0.2 cm; 6, 0.5 to 1 cm young panicle; 7, 3 to 5 cm young panicle; 8, longer than 10 cm young panicle; 9, stamen; 10, pistil; 11, shoot at 1 d after germination; 12, 3-week-old shoot; 13, 3-week-old etiolated shoot.

Figure 5.

Overexpression of OsmiR156 in rice. A, Schematic diagram of OsmiR156 overexpression construct. UBI, Maize ubiquitin gene promoter; Hpt, hygromycin resistance gene. B, PAGE RNA gel-blot analysis of miR156 in the leaves of transgenic plants at tillering stage. The exposure time of blot was optimal for the signal of overexpressed transgene but not long enough to show the signal in wild type (WT). Mb, OsmiR156b overexpression; Mh, OsmiR156h overexpression. C, Reduced plant height and increased number of tillers in Mb and Mh transgenic plants. The scale bar is 10 cm. D, Number of spikelets and grains per panicle in the transgenic plants and number of secondary branches per panicle in the transgenic plants.

Figure 6.

Transcript levels of OsmiR156 and target genes in the transgenic plants. A, Semiquantitative RT-PCR of nine putative target genes of OsmiR156 in flag leaves and young panicles of the OsmiR156b (Mb)- and OsmiR156h (Mh)-overexpressing plants. The primers with amplicons spanning the complementary sites were used. The rice Actin1 gene was amplified as the internal control. B, Real-time PCR analysis of four putative target genes of OsmiR156. The relative expression levels were calibrated based on the leaf sample of wild type. C, Transcript levels of two OsmiR156-targeted genes in the young leaves of transgenic plants overexpressing the target genes. DNA fragments downstream of the targeting sites were used as probes. The target gene-overexpressed plants are indicated by diamonds. The uncleaved and cleaved transcripts (bands) are indicated by arrows.