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

Zhu QH, Upadhyaya NM, Gubler F, Helliwell CA - BMC Plant Biol. (2009)

Bottom Line: However SNB expression was not reduced in the miR172b over-expression plants.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. qianhao.zhu@csiro.au

ABSTRACT

Background: Regulation 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.

Results: Analysis 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.

Conclusions: The 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.

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Analysis of miR172-mediated cleavage of target genes. 5' RACE was used to map the miR172-mediated cleavage sites in the predicted targets. The expected cleavage site is indicated by an arrow. Nucleotides that differ among miR172 family members or their targets are shown in bold italic. The cleavage frequencies (number of clones with the expected cleavage site/total number of clones sequenced) detected in the indicated tissues are shown to the right of the sequence alignment. BP: booting panicle. nd: no RACE product detected.
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Figure 3: Analysis of miR172-mediated cleavage of target genes. 5' RACE was used to map the miR172-mediated cleavage sites in the predicted targets. The expected cleavage site is indicated by an arrow. Nucleotides that differ among miR172 family members or their targets are shown in bold italic. The cleavage frequencies (number of clones with the expected cleavage site/total number of clones sequenced) detected in the indicated tissues are shown to the right of the sequence alignment. BP: booting panicle. nd: no RACE product detected.

Mentions: miR172 has been shown to cleave AP2 and AP2-like target mRNAs in Arabidopsis [13,14,21] and maize [15,17], but is thought to act predominantly through translational repression [13-15]. To determine whether the five putative targets of miR172 in rice are cleaved by miR172, 5' rapid amplification of cDNA ends (RACE) analysis was performed using RNA isolated from two-leaf stage shoots, 1-10 DAF grains and booting panicles (BP). Cleavage of Os04g55560 was detected in a mixed sample of shoot and grain as well as in booting panicles; cleavage of Os06g43220 was only detected in the mixed sample with a low frequency (most likely contributed by young seedlings as accumulation of miR172 was below the detection limit in 10 DAF grains); and cleavage of SNB was only detected in booting panicles. No cleavage was detected for Os03g60430 or Os05g03040 in any of the samples analyzed (Figure 3). These results suggested tissue- or cell-type-specific expression of miR172 and/or its target genes.


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

Zhu QH, Upadhyaya NM, Gubler F, Helliwell CA - BMC Plant Biol. (2009)

Analysis of miR172-mediated cleavage of target genes. 5' RACE was used to map the miR172-mediated cleavage sites in the predicted targets. The expected cleavage site is indicated by an arrow. Nucleotides that differ among miR172 family members or their targets are shown in bold italic. The cleavage frequencies (number of clones with the expected cleavage site/total number of clones sequenced) detected in the indicated tissues are shown to the right of the sequence alignment. BP: booting panicle. nd: no RACE product detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803185&req=5

Figure 3: Analysis of miR172-mediated cleavage of target genes. 5' RACE was used to map the miR172-mediated cleavage sites in the predicted targets. The expected cleavage site is indicated by an arrow. Nucleotides that differ among miR172 family members or their targets are shown in bold italic. The cleavage frequencies (number of clones with the expected cleavage site/total number of clones sequenced) detected in the indicated tissues are shown to the right of the sequence alignment. BP: booting panicle. nd: no RACE product detected.
Mentions: miR172 has been shown to cleave AP2 and AP2-like target mRNAs in Arabidopsis [13,14,21] and maize [15,17], but is thought to act predominantly through translational repression [13-15]. To determine whether the five putative targets of miR172 in rice are cleaved by miR172, 5' rapid amplification of cDNA ends (RACE) analysis was performed using RNA isolated from two-leaf stage shoots, 1-10 DAF grains and booting panicles (BP). Cleavage of Os04g55560 was detected in a mixed sample of shoot and grain as well as in booting panicles; cleavage of Os06g43220 was only detected in the mixed sample with a low frequency (most likely contributed by young seedlings as accumulation of miR172 was below the detection limit in 10 DAF grains); and cleavage of SNB was only detected in booting panicles. No cleavage was detected for Os03g60430 or Os05g03040 in any of the samples analyzed (Figure 3). These results suggested tissue- or cell-type-specific expression of miR172 and/or its target genes.

Bottom Line: However SNB expression was not reduced in the miR172b over-expression plants.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. qianhao.zhu@csiro.au

ABSTRACT

Background: Regulation 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.

Results: Analysis 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus