Plant Physiol.-2006-Xie-280-93.docx

Genomic Organization, Differential Expression, and Interaction of SQUAMOSA Promoter-Binding-Like Transcription Factors and microRNA156 in RiceKabin Xie, Congqing Wu, and Lizhong Xiong*Plant Physiology, September 2006, Vol. 142, pp. 280293National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(Wuhan), Huazhong Agricultural University, Wuhan 430070, ChinaBACKGROUND1. SQUAMOSA (SQUA) promoter-binding-like (SPL) proteins contain a highly conserved DNA-binding domain (SQUA promoter-binding protein SBP domain.2. SBP domain features a novel zinc nger motif that contains two zinc-binding sites assembled as Cys-Cys-His-Cys and Cys-Cys-Cys-His.3. In the Arabidopsis genome, 16 putative SPL genes contained the SBP domain.4. 10 of 16 SPL genes were regulated by miRNA genes of the miRNA156 family in Arabidopisis.5. Here the report on the analyses of SPL and miR156 for their genomic organization, gene structures, motif composition, and expression levels in various tissues and organs of rice.RESULTS1. Identication of OsSPL and OsmiR156 Genes in the Rice Genome1.1 11 of 19 OsSPLs contained sequences that are complementary to the OsmiR156 mature sequence , with one mismatch at the 14th nucleotide. (Fig. 1A)1.2 At least one OsSPL gene contains the sequence that is complementary to the mature sequence of miR156. (Fig. 1B)2. Phylogenetic Analysis of the OsSPL and OsmiR156 Gene Families3.Diverse Exon-Intron Structure and Motif Composition in OsSPL Family4. Differential Expression Proles of OsSPL and OsmiR156 GenesThe expression patterns of OsSPL genes can be classied into three types according to their expression patterns.Semiquantitative RT-PCRThe amplifying the region covering the targeting site (labeled with an asterisk).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 shootReal-time quantitative PCR analysis of four OsSPL genes (Fig. 4B)PAGE RNA gel-blot analysisPAGE RNA gel-blot analysis also showed that the mature OsmiR156 had strong expression in young shoot, etiolated shoot, and seedling leaves, weak expression in root, stamen, and pistil, and undetectable expression level in stem and young panicles (Fig. 4C).5. Overexpression of OsmiR156 in Rice Resulted in Abnormal Growth and DevelopmentFigure 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.6. Differential Interaction between OsmiR156 and Target OsSPL Genes 6.1 OsSPL3, OsSPL7, and OsSPL11 had no change of expression level in either tissue. 6.2 Three genes(OsSPL2, OsSPL12, and OsSPL13) had decreased mRNA levels in the ag leaves of transgenic plants.6.3 Two genes (OsSPL16 and OSPL18) had obviously decreased mRNA levels in the panicles.6.4 OsSPL14 had decreased mRNA levels in both ag leaves and panicles.RNA gel-blot analysisDifferent target OsSPL genes may be differentially regulated by OsmiR156.DISCUSSIONDiverse Tempospatial Expression Patterns of OsSPL GenesThe transcript levels of all OsSPL genes in 13 different tissues of rice suggest that OsSPL genesmight have different expression patterns.Function and Interaction of OsmiR156 and OsSPLsMost miRNAs regulate more than one targe