Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa)

BackgroundRegulation of gene expression by microRNAs (miRNAs) plays a crucial role in many developmental and physiological processes in plants. miRNAs act to repress expression of their target genes via mRNA cleavage or translational repression. Dozens of miRNA families have been identified in rice, 21 of which are conserved between rice and Arabidopsis. miR172 is a conserved miRNA family which has been shown to regulate expression of APETALA2 (AP2)-like transcription factors in Arabidopsis and maize. The rice genome encodes five AP2-like genes predicted to be targets of miR172. To determine whether these rice AP2-like genes are regulated by miR172 and investigate the function of the target genes, we studied the effect of over-expressing two members of the miR172 family on rice plant development.ResultsAnalysis of miR172 expression showed that it is most highly expressed in late vegetative stages and developing panicles. Analyses of expression of three miR172 targets showed that SUPERNUMERARY BRACT (SNB) and Os03g60430 have high expression in developing panicles. Expression of miR172 was not inversely correlated with expression of its targets although miR172-mediated cleavage of SNB was detected by 5' rapid amplification of cDNA ends (RACE). Over-expression of miR172b in rice delayed the transition from spikelet meristem to floral meristem, and resulted in floral and seed developmental defects, including changes to the number and identity of floral organs, lower fertility and reduced seed weight. Plants over-expressing miR172b not only phenocopied the T-DNA insertion mutant of SNB but showed additional defects in floret development not seen in the snb mutant. However SNB expression was not reduced in the miR172b over-expression plants.ConclusionsThe phenotypes resulting from over-expression of miR172b suggests it represses SNB and at least one of the other miR172 targets, most likely Os03g60430, indicating roles for other AP2-like genes in rice floret development. miR172 and the AP2-like genes had overlapping expression patterns in rice and their expression did not show an obvious negative correlation. There was not a uniform decrease in the expression of the AP2-like miR172 target mRNAs in the miR172b over-expression plants. These observations are consistent with miR172 functioning via translational repression or with expression of the AP2-like genes being regulated by a negative feedback loop.

[1]  S. Hake,et al.  Floral meristem initiation and meristem cell fate are regulated by the maize AP2 genes ids1 and sid1 , 2008, Development.

[2]  E. Dennis,et al.  Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice , 2006, Theoretical and Applied Genetics.

[3]  Roberto Tuberosa,et al.  Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize , 2007, Proceedings of the National Academy of Sciences.

[4]  Ryan D. Morin,et al.  Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. , 2008, Genome research.

[5]  K. Shimamoto,et al.  FRIZZY PANICLE is required to prevent the formation of axillary meristems and to establish floral meristem identity in rice spikelets , 2003, Development.

[6]  Xuemei Chen,et al.  Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana , 2006, Plant Molecular Biology.

[7]  R. Sunkar,et al.  The role of microRNAs and other endogenous small RNAs in plant stress responses. , 2008, Biochimica et biophysica acta.

[8]  Xuemei Chen,et al.  A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development , 2004, Science.

[9]  Yun Zheng,et al.  Identification of novel and candidate miRNAs in rice by high throughput sequencing , 2008, BMC Plant Biology.

[10]  Mingsheng Chen,et al.  The phylogeny and expression pattern of APETALA2-like genes in rice. , 2007, Journal of genetics and genomics = Yi chuan xue bao.

[11]  Xuemei Chen,et al.  miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. , 2007, The Plant journal : for cell and molecular biology.

[12]  H. Vaucheret,et al.  Functions of microRNAs and related small RNAs in plants , 2006, Nature Genetics.

[13]  N. Chua,et al.  The GIGANTEA-Regulated MicroRNA172 Mediates Photoperiodic Flowering Independent of CONSTANS in Arabidopsis[W][OA] , 2007, The Plant Cell Online.

[14]  Javier F. Palatnik,et al.  Specific effects of microRNAs on the plant transcriptome. , 2005, Developmental cell.

[15]  C. Helliwell,et al.  A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. , 2008, Genome research.

[16]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[17]  Hong-Wei Xue,et al.  Characterization and expression profiles of miRNAs in rice seeds , 2008, Nucleic acids research.

[18]  S. Moose,et al.  microRNA172 down-regulates glossy15 to promote vegetative phase change in maize. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Ramanjulu Sunkar,et al.  Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. , 2007, Trends in plant science.

[20]  S. Hake,et al.  The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1 , 2007, Nature Genetics.

[21]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[22]  Hajime Sakai,et al.  Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238. , 2003, The Plant Cell Online.

[23]  B. Gill,et al.  Molecular Characterization of the Major Wheat Domestication Gene Q , 2006, Genetics.

[24]  E. Dennis,et al.  Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L) , 2003, BMC Plant Biology.

[25]  B. Reinhart,et al.  MicroRNAs in plants. , 2002, Genes & development.

[26]  Bin Liu,et al.  Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs) , 2008, Proceedings of the National Academy of Sciences.

[27]  J. Bowman,et al.  Genetic interactions among floral homeotic genes of Arabidopsis. , 1991, Development.

[28]  D. Bartel,et al.  MicroRNAS and their regulatory roles in plants. , 2006, Annual review of plant biology.

[29]  P. Waterhouse,et al.  Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers , 2000 .

[30]  S. Luo,et al.  Global identification of microRNA–target RNA pairs by parallel analysis of RNA ends , 2008, Nature Biotechnology.

[31]  G. An,et al.  The rice heterochronic gene SUPERNUMERARY BRACT regulates the transition from spikelet meristem to floral meristem. , 2006, The Plant journal : for cell and molecular biology.

[32]  S. Hake,et al.  The Control of Spikelet Meristem Identity by the branched silkless1 Gene in Maize , 2002, Science.

[33]  S. Hake,et al.  The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet1. , 1998, Genes & development.