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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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Changes in the number of floral organs and floral identity in plants over-expressing miR172b. A, A wild-type floret with lemma and palea removed to show lodicule (only one is seen which is indicated by a red arrow), six anthers (indicated by blue arrows), one carpel (indicated by a red *) and two stigmas (indicated by light pink arrows). B, SEM of the basal part of a wild-type spikelet showing the morphology and surface features of a lodicule. C and D, Close-ups of the lodicule in the white and blue boxed regions in B, respectively. E to T, Images of floret or floral organs of plants over-expressing miR172b. E, Two swollen lodicules fused together. F and G, Two enlarged lodicules fused together to form a cup-like structure. H, Multiple lodicules located at the same whorl. I and J, Multiple lodicules located at two whorls with elongated lodicules at the outer whorl. K, All four lodicules are elongated and show distinct features in the middle and flanking regions at the base. L, A close-up of the white boxed region shown in K. M, An elongated lodicule (tip of the lodicule is indicated by a yellow arrow) fused with an anther (indicated by a blue arrow). N, Lodicule base and anther top organ replaced carpel and completely separated two stigmas that showed flat style. O, A close-up of the boxed portion in N. P, Conversion part of the stigma into an anther. Q, Floret with three stigmas. R, Floret with two carpels (indicated by red *). S and T, Two florets developed within a single spikelet and one of them (left side one) always with incomplete floral organs. White arrows indicate lemma or palea. To show the internal floral organs, both lemma and palea (for A, B, E, G to J and N to R) or one of them (for F, K, S and T) has been removed. EG: empty glume, FL: filament. Bars in A, F, H to J, and P to T are 1 mm. Bars in B, E and G are 200 μm. Bars in C, D and L are 42 μm. Bars in K, M to O are 420 μm.
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Figure 6: Changes in the number of floral organs and floral identity in plants over-expressing miR172b. A, A wild-type floret with lemma and palea removed to show lodicule (only one is seen which is indicated by a red arrow), six anthers (indicated by blue arrows), one carpel (indicated by a red *) and two stigmas (indicated by light pink arrows). B, SEM of the basal part of a wild-type spikelet showing the morphology and surface features of a lodicule. C and D, Close-ups of the lodicule in the white and blue boxed regions in B, respectively. E to T, Images of floret or floral organs of plants over-expressing miR172b. E, Two swollen lodicules fused together. F and G, Two enlarged lodicules fused together to form a cup-like structure. H, Multiple lodicules located at the same whorl. I and J, Multiple lodicules located at two whorls with elongated lodicules at the outer whorl. K, All four lodicules are elongated and show distinct features in the middle and flanking regions at the base. L, A close-up of the white boxed region shown in K. M, An elongated lodicule (tip of the lodicule is indicated by a yellow arrow) fused with an anther (indicated by a blue arrow). N, Lodicule base and anther top organ replaced carpel and completely separated two stigmas that showed flat style. O, A close-up of the boxed portion in N. P, Conversion part of the stigma into an anther. Q, Floret with three stigmas. R, Floret with two carpels (indicated by red *). S and T, Two florets developed within a single spikelet and one of them (left side one) always with incomplete floral organs. White arrows indicate lemma or palea. To show the internal floral organs, both lemma and palea (for A, B, E, G to J and N to R) or one of them (for F, K, S and T) has been removed. EG: empty glume, FL: filament. Bars in A, F, H to J, and P to T are 1 mm. Bars in B, E and G are 200 μm. Bars in C, D and L are 42 μm. Bars in K, M to O are 420 μm.

Mentions: The unit comprising lemma, palea and floral organs including two lodicules on the lemma side, six anthers and a carpel with two stigmas is called floret (Figure 6A). Wild-type lodicules have a wide base, a rough surface and a narrow apex (Figure 6B, C, D). When flowering, the floret opens due to swelling of the lodicules, and closes after a few minutes (depending on temperature and humidity) due to shrinking of the lodicules. In plants over-expressing miR172b, florets with five or more layers of lemma and palea could not open due to the tightly closed lemma and palea. Florets that did not completely close up after flowering had altered numbers and/or morphology of lodicules. One (due to fusion of two lodicules; Figure 6E, F, G) to as many as eight lodicules were observed (Table 2). Multiple lodicules were arranged in one (Figure 6H to 6K) or two (Figure 6I, J) whorls, with similar surface features to those of wild-type but swollen (Figure 6H), or elongated significantly and converted into a structure similar to the palea marginal region (Figure 6I, J, K). In the case of two whorls of lodicules, usually only the lodicule in the outer whorl was converted (Figure 6J). In the converted lodicules, two edges of the base section retained their original identity (Figure 6L), resulting in a mosaic floral organ. The most frequently observed mosaic floral organ was a lodicule base with an anther fused to the elongated lodicule apex (Figure 6M). Occasionally, a mosaic organ with a lodicule base and an anther top was observed at the innermost whorl of the floret, in which the mosaic organ replaced the carpel and the identities of two stigmas were also converted (Figure 6N, O). In some florets, a stigma was partially converted into an anther (Figure 6P). These results suggest that timing and/or positioning of the floral organ meristems are interrupted by over-expression of miR172b, indicating that a proper expression of miR172 target genes is important in specification of floral organ identities.


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)

Changes in the number of floral organs and floral identity in plants over-expressing miR172b. A, A wild-type floret with lemma and palea removed to show lodicule (only one is seen which is indicated by a red arrow), six anthers (indicated by blue arrows), one carpel (indicated by a red *) and two stigmas (indicated by light pink arrows). B, SEM of the basal part of a wild-type spikelet showing the morphology and surface features of a lodicule. C and D, Close-ups of the lodicule in the white and blue boxed regions in B, respectively. E to T, Images of floret or floral organs of plants over-expressing miR172b. E, Two swollen lodicules fused together. F and G, Two enlarged lodicules fused together to form a cup-like structure. H, Multiple lodicules located at the same whorl. I and J, Multiple lodicules located at two whorls with elongated lodicules at the outer whorl. K, All four lodicules are elongated and show distinct features in the middle and flanking regions at the base. L, A close-up of the white boxed region shown in K. M, An elongated lodicule (tip of the lodicule is indicated by a yellow arrow) fused with an anther (indicated by a blue arrow). N, Lodicule base and anther top organ replaced carpel and completely separated two stigmas that showed flat style. O, A close-up of the boxed portion in N. P, Conversion part of the stigma into an anther. Q, Floret with three stigmas. R, Floret with two carpels (indicated by red *). S and T, Two florets developed within a single spikelet and one of them (left side one) always with incomplete floral organs. White arrows indicate lemma or palea. To show the internal floral organs, both lemma and palea (for A, B, E, G to J and N to R) or one of them (for F, K, S and T) has been removed. EG: empty glume, FL: filament. Bars in A, F, H to J, and P to T are 1 mm. Bars in B, E and G are 200 μm. Bars in C, D and L are 42 μm. Bars in K, M to O are 420 μm.
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: Changes in the number of floral organs and floral identity in plants over-expressing miR172b. A, A wild-type floret with lemma and palea removed to show lodicule (only one is seen which is indicated by a red arrow), six anthers (indicated by blue arrows), one carpel (indicated by a red *) and two stigmas (indicated by light pink arrows). B, SEM of the basal part of a wild-type spikelet showing the morphology and surface features of a lodicule. C and D, Close-ups of the lodicule in the white and blue boxed regions in B, respectively. E to T, Images of floret or floral organs of plants over-expressing miR172b. E, Two swollen lodicules fused together. F and G, Two enlarged lodicules fused together to form a cup-like structure. H, Multiple lodicules located at the same whorl. I and J, Multiple lodicules located at two whorls with elongated lodicules at the outer whorl. K, All four lodicules are elongated and show distinct features in the middle and flanking regions at the base. L, A close-up of the white boxed region shown in K. M, An elongated lodicule (tip of the lodicule is indicated by a yellow arrow) fused with an anther (indicated by a blue arrow). N, Lodicule base and anther top organ replaced carpel and completely separated two stigmas that showed flat style. O, A close-up of the boxed portion in N. P, Conversion part of the stigma into an anther. Q, Floret with three stigmas. R, Floret with two carpels (indicated by red *). S and T, Two florets developed within a single spikelet and one of them (left side one) always with incomplete floral organs. White arrows indicate lemma or palea. To show the internal floral organs, both lemma and palea (for A, B, E, G to J and N to R) or one of them (for F, K, S and T) has been removed. EG: empty glume, FL: filament. Bars in A, F, H to J, and P to T are 1 mm. Bars in B, E and G are 200 μm. Bars in C, D and L are 42 μm. Bars in K, M to O are 420 μm.
Mentions: The unit comprising lemma, palea and floral organs including two lodicules on the lemma side, six anthers and a carpel with two stigmas is called floret (Figure 6A). Wild-type lodicules have a wide base, a rough surface and a narrow apex (Figure 6B, C, D). When flowering, the floret opens due to swelling of the lodicules, and closes after a few minutes (depending on temperature and humidity) due to shrinking of the lodicules. In plants over-expressing miR172b, florets with five or more layers of lemma and palea could not open due to the tightly closed lemma and palea. Florets that did not completely close up after flowering had altered numbers and/or morphology of lodicules. One (due to fusion of two lodicules; Figure 6E, F, G) to as many as eight lodicules were observed (Table 2). Multiple lodicules were arranged in one (Figure 6H to 6K) or two (Figure 6I, J) whorls, with similar surface features to those of wild-type but swollen (Figure 6H), or elongated significantly and converted into a structure similar to the palea marginal region (Figure 6I, J, K). In the case of two whorls of lodicules, usually only the lodicule in the outer whorl was converted (Figure 6J). In the converted lodicules, two edges of the base section retained their original identity (Figure 6L), resulting in a mosaic floral organ. The most frequently observed mosaic floral organ was a lodicule base with an anther fused to the elongated lodicule apex (Figure 6M). Occasionally, a mosaic organ with a lodicule base and an anther top was observed at the innermost whorl of the floret, in which the mosaic organ replaced the carpel and the identities of two stigmas were also converted (Figure 6N, O). In some florets, a stigma was partially converted into an anther (Figure 6P). These results suggest that timing and/or positioning of the floral organ meristems are interrupted by over-expression of miR172b, indicating that a proper expression of miR172 target genes is important in specification of floral organ identities.

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