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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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qRT-PCR analyses of miR172 target genes in wild-type plants. A primer pair spanning the miR172 target site was used to quantify expression of the uncleaved target mRNAs. For each gene, relative fold expression difference is shown by using the expression level detected in flag leaf as the reference. Error bars represent standard deviation of the expression ratio. 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. ≤ 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: embryo. En: endosperm.
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Figure 2: qRT-PCR analyses of miR172 target genes in wild-type plants. A primer pair spanning the miR172 target site was used to quantify expression of the uncleaved target mRNAs. For each gene, relative fold expression difference is shown by using the expression level detected in flag leaf as the reference. Error bars represent standard deviation of the expression ratio. 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. ≤ 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: embryo. En: endosperm.

Mentions: The abundance of intact transcripts of miR172 target genes was analyzed by qRT-PCR using primer pairs spanning the miR172 cleavage sites. Expression of SNB (Os07g13170) was highest in developing panicles (<4 cm in length), in which differentiation of the spikelet and floral organs is progressing; expression of SNB was also high in roots from 10-leaf plants (Figure 2A). Os03g60430 was highly expressed in developing panicles and also in young seedlings (2L-S) (Figure 2B). In contrast expression of Os05g03040 was highest in young seedlings and roots (Figure 2C). All these three genes had a very low expression level in embryo, endosperm and pericarp of 10 DAF grains (Figure 2A, B, C). Expression of SNB and Os03g60430 showed an inverse correlation with the abundance of miR172 in two-leaf shoots, leaf four and leaf ten, but generally the expression of miR172 was not inversely correlated with the expression of its targets in the tissues analyzed (Figure 2A, B, C). We were unable to specifically amplify Os06g43220 even though several primer combinations were tried; this could be a result of very low expression (supported by the relatively low number of ESTs found in both japonica and indica rice; data not shown). We were unable to quantify Os04g55560 expression as the gene-specific product was always accompanied by a non-specific product. These expression profiles support previous results showing that SNB has a role in controlling spikelet determinacy and floret development [24], and also suggest that Os03g60430 could play a role in floret development.


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)

qRT-PCR analyses of miR172 target genes in wild-type plants. A primer pair spanning the miR172 target site was used to quantify expression of the uncleaved target mRNAs. For each gene, relative fold expression difference is shown by using the expression level detected in flag leaf as the reference. Error bars represent standard deviation of the expression ratio. 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. ≤ 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: embryo. En: endosperm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: qRT-PCR analyses of miR172 target genes in wild-type plants. A primer pair spanning the miR172 target site was used to quantify expression of the uncleaved target mRNAs. For each gene, relative fold expression difference is shown by using the expression level detected in flag leaf as the reference. Error bars represent standard deviation of the expression ratio. 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. ≤ 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: embryo. En: endosperm.
Mentions: The abundance of intact transcripts of miR172 target genes was analyzed by qRT-PCR using primer pairs spanning the miR172 cleavage sites. Expression of SNB (Os07g13170) was highest in developing panicles (<4 cm in length), in which differentiation of the spikelet and floral organs is progressing; expression of SNB was also high in roots from 10-leaf plants (Figure 2A). Os03g60430 was highly expressed in developing panicles and also in young seedlings (2L-S) (Figure 2B). In contrast expression of Os05g03040 was highest in young seedlings and roots (Figure 2C). All these three genes had a very low expression level in embryo, endosperm and pericarp of 10 DAF grains (Figure 2A, B, C). Expression of SNB and Os03g60430 showed an inverse correlation with the abundance of miR172 in two-leaf shoots, leaf four and leaf ten, but generally the expression of miR172 was not inversely correlated with the expression of its targets in the tissues analyzed (Figure 2A, B, C). We were unable to specifically amplify Os06g43220 even though several primer combinations were tried; this could be a result of very low expression (supported by the relatively low number of ESTs found in both japonica and indica rice; data not shown). We were unable to quantify Os04g55560 expression as the gene-specific product was always accompanied by a non-specific product. These expression profiles support previous results showing that SNB has a role in controlling spikelet determinacy and floret development [24], and also suggest that Os03g60430 could play a role in floret development.

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