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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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RNA gel blot analysis of accumulation of miR172 in wild-type plants. A, Accumulation of miR172 in vegetative tissues. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA: shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf. B, Accumulation of miR172 in reproductive tissues and grains. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4 cm, respectively. BP: booting panicle. Em, En and Pe: embryo, endosperm and pericarp of 10 DAF grains, respectively.
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Figure 1: RNA gel blot analysis of accumulation of miR172 in wild-type plants. A, Accumulation of miR172 in vegetative tissues. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA: shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf. B, Accumulation of miR172 in reproductive tissues and grains. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4 cm, respectively. BP: booting panicle. Em, En and Pe: embryo, endosperm and pericarp of 10 DAF grains, respectively.

Mentions: To determine where miR172 and its target transcripts are expressed during rice development, we analyzed miR172 expression by RNA gel blot and expression of the AP2-like target mRNAs by qRT-PCR in various tissues. The mature miR172a-d sequences differ only in their 5' and 3' bases and therefore hybridization with a miR172a probe is likely to detect expression of all mature miR172 sequences. In wild-type plants, miR172 expression varied considerably between organs and developmental stages. Mature miR172 accumulation increased significantly in leaves but not in roots as plants grew, reaching a maximum in the flag leaf (Figure 1A). Similar expression patterns of miR172 have also been observed in vegetative tissues of Arabidopsis and maize [13,17], suggesting that miR172 has a conserved role during vegetative development. In reproductive tissues, miR172 was consistently expressed although its abundance reduced gradually during panicle development (Figure 1B). Expression of miR172 was below the detection limit in 10 DAF (days-after-fertilization) grains (Figure 1B). Higher expression of miR172 in later stage vegetative tissues and developing young panicles is consistent with a role in regulating the timing of floret initiation and development in rice.


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)

RNA gel blot analysis of accumulation of miR172 in wild-type plants. A, Accumulation of miR172 in vegetative tissues. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA: shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf. B, Accumulation of miR172 in reproductive tissues and grains. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4 cm, respectively. BP: booting panicle. Em, En and Pe: embryo, endosperm and pericarp of 10 DAF grains, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: RNA gel blot analysis of accumulation of miR172 in wild-type plants. A, Accumulation of miR172 in vegetative tissues. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA: shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf. B, Accumulation of miR172 in reproductive tissues and grains. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4 cm, respectively. BP: booting panicle. Em, En and Pe: embryo, endosperm and pericarp of 10 DAF grains, respectively.
Mentions: To determine where miR172 and its target transcripts are expressed during rice development, we analyzed miR172 expression by RNA gel blot and expression of the AP2-like target mRNAs by qRT-PCR in various tissues. The mature miR172a-d sequences differ only in their 5' and 3' bases and therefore hybridization with a miR172a probe is likely to detect expression of all mature miR172 sequences. In wild-type plants, miR172 expression varied considerably between organs and developmental stages. Mature miR172 accumulation increased significantly in leaves but not in roots as plants grew, reaching a maximum in the flag leaf (Figure 1A). Similar expression patterns of miR172 have also been observed in vegetative tissues of Arabidopsis and maize [13,17], suggesting that miR172 has a conserved role during vegetative development. In reproductive tissues, miR172 was consistently expressed although its abundance reduced gradually during panicle development (Figure 1B). Expression of miR172 was below the detection limit in 10 DAF (days-after-fertilization) grains (Figure 1B). Higher expression of miR172 in later stage vegetative tissues and developing young panicles is consistent with a role in regulating the timing of floret initiation and development in rice.

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