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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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Phenotypes of spikelets and mature seeds of plants over-expressing miR172b. A, A wild-type spikelet comprising a single floret enclosed by lemma and palea, a pair of rudimentary glumes (indicated by pink arrow heads) and a pair of empty glumes (indicated by white arrow heads). B to I, Individual spikelets from plants over-expressing miR172b. All of these spikelets do not have empty glumes but have multiple bract-like structures (top pair indicated by a pair of pink arrow heads). B, Fertile spikelet with a pair of lemma and palea (indicated by red *, same for C to G). C, Sterile spikelet with two pairs of lemma and palea. D, Sterile spikelet with multiple pairs of lemma and palea. The boxed part still contains multiple layers of lemma and palea. E, Sterile spikelet with a pair of twisted lemma and palea. F, Sterile spikelet with a normal-looking lemma or palea and a degenerated lemma or palea (indicated by blue *, same for H). G, Sterile spikelet with elongated lemma or palea. H, Sterile spikelet with multiple layers of degenerated lemma or palea. I, Sterile spikelet without floret. J, Scanning electron micrograph (SEM) of wild-type spikelet to show the surface features of empty glumes and rudimentary glumes. K, SEM of the bract-like structures of a spikelet from a miR172b over-expression plant. Green arrows in J and K indicate trichomes. L, Part of a wild-type panicle with normal spikelets. M, Part of a panicle from a miR172b over-expression plant with a strongly altered spikelet phenotype. N to P, Part of panicles from a miR172b over-expression plant with a moderately altered spikelet phenotype. White arrows in L to P indicate individual spikelets. Spikelets indicated by pink arrows in O and P represent spikelets with degenerated lemma or palea. Q, Wild-type mature seed. R to T, Mature seeds from plants over-expressing miR172b showing naked single grain (R and S) or double grains (T). EG: empty glume. LE: lemma. PA: palea. RG: rudimentary glume. Bars in A to I, and Q to T are 1 mm. Bars in J and K are 100 μm.
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Figure 5: Phenotypes of spikelets and mature seeds of plants over-expressing miR172b. A, A wild-type spikelet comprising a single floret enclosed by lemma and palea, a pair of rudimentary glumes (indicated by pink arrow heads) and a pair of empty glumes (indicated by white arrow heads). B to I, Individual spikelets from plants over-expressing miR172b. All of these spikelets do not have empty glumes but have multiple bract-like structures (top pair indicated by a pair of pink arrow heads). B, Fertile spikelet with a pair of lemma and palea (indicated by red *, same for C to G). C, Sterile spikelet with two pairs of lemma and palea. D, Sterile spikelet with multiple pairs of lemma and palea. The boxed part still contains multiple layers of lemma and palea. E, Sterile spikelet with a pair of twisted lemma and palea. F, Sterile spikelet with a normal-looking lemma or palea and a degenerated lemma or palea (indicated by blue *, same for H). G, Sterile spikelet with elongated lemma or palea. H, Sterile spikelet with multiple layers of degenerated lemma or palea. I, Sterile spikelet without floret. J, Scanning electron micrograph (SEM) of wild-type spikelet to show the surface features of empty glumes and rudimentary glumes. K, SEM of the bract-like structures of a spikelet from a miR172b over-expression plant. Green arrows in J and K indicate trichomes. L, Part of a wild-type panicle with normal spikelets. M, Part of a panicle from a miR172b over-expression plant with a strongly altered spikelet phenotype. N to P, Part of panicles from a miR172b over-expression plant with a moderately altered spikelet phenotype. White arrows in L to P indicate individual spikelets. Spikelets indicated by pink arrows in O and P represent spikelets with degenerated lemma or palea. Q, Wild-type mature seed. R to T, Mature seeds from plants over-expressing miR172b showing naked single grain (R and S) or double grains (T). EG: empty glume. LE: lemma. PA: palea. RG: rudimentary glume. Bars in A to I, and Q to T are 1 mm. Bars in J and K are 100 μm.

Mentions: We generated transgenic plants expressing the stem-loop precursors of miR172a and miR172b, transcribed by the maize ubiquitin promoter. Elevated levels of miR172 were detected in these miR172 over-expression plants, particularly in plants transformed with pre-MIR172b (Figure 4). Transgenic plants over-expressing miR172b showed normal vegetative growth and heading time, but the inflorescence (panicle) of the transformed plants was smaller, producing the half number of primary branches of the untransformed wild-type (Table 1). A wild-type spikelet consists of a single floret and two subtending pairs of bract-like structures - a pair of rudimentary glumes and a pair of empty glumes (Figure 5A, J). Spikelets of plants over-expressing miR172b were generated from primary or secondary branches as in wild-type, but the majority of spikelets were abnormal and showed variable defects in floral organs. The common phenotypes of the mutated spikelets were that more than two, and in extreme cases as many as 20, bract-like structures were generated before transition to floral development (Figure 5B to 5H); in some cases no obvious floral organs were produced (Figure 5I). The majority of these spikelets lacked a pair of empty glumes (compare Figure 5A with Figure 5B to 5H; Table 2). Scanning electron microscopy (SEM) showed that the lower part of the rudimentary glumes in the wild-type plant had round projections and small trichomes (Figure 5J). In the miR172b over-expression plants, similar round projections coated the surface of the bract-like structures though fewer trichomes were seen (Figure 5K), suggesting that the additional bract-like structures in the miR172b over-expression lines have the same identity as rudimentary glumes. This result suggests that reproductive development of plants over-expressing miR172b was not affected until the formation of the spikelet meristem, but the transition from spikelet meristem to floral meristem was delayed, leading to the reiteration of bract-like structures.


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)

Phenotypes of spikelets and mature seeds of plants over-expressing miR172b. A, A wild-type spikelet comprising a single floret enclosed by lemma and palea, a pair of rudimentary glumes (indicated by pink arrow heads) and a pair of empty glumes (indicated by white arrow heads). B to I, Individual spikelets from plants over-expressing miR172b. All of these spikelets do not have empty glumes but have multiple bract-like structures (top pair indicated by a pair of pink arrow heads). B, Fertile spikelet with a pair of lemma and palea (indicated by red *, same for C to G). C, Sterile spikelet with two pairs of lemma and palea. D, Sterile spikelet with multiple pairs of lemma and palea. The boxed part still contains multiple layers of lemma and palea. E, Sterile spikelet with a pair of twisted lemma and palea. F, Sterile spikelet with a normal-looking lemma or palea and a degenerated lemma or palea (indicated by blue *, same for H). G, Sterile spikelet with elongated lemma or palea. H, Sterile spikelet with multiple layers of degenerated lemma or palea. I, Sterile spikelet without floret. J, Scanning electron micrograph (SEM) of wild-type spikelet to show the surface features of empty glumes and rudimentary glumes. K, SEM of the bract-like structures of a spikelet from a miR172b over-expression plant. Green arrows in J and K indicate trichomes. L, Part of a wild-type panicle with normal spikelets. M, Part of a panicle from a miR172b over-expression plant with a strongly altered spikelet phenotype. N to P, Part of panicles from a miR172b over-expression plant with a moderately altered spikelet phenotype. White arrows in L to P indicate individual spikelets. Spikelets indicated by pink arrows in O and P represent spikelets with degenerated lemma or palea. Q, Wild-type mature seed. R to T, Mature seeds from plants over-expressing miR172b showing naked single grain (R and S) or double grains (T). EG: empty glume. LE: lemma. PA: palea. RG: rudimentary glume. Bars in A to I, and Q to T are 1 mm. Bars in J and K are 100 μm.
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Figure 5: Phenotypes of spikelets and mature seeds of plants over-expressing miR172b. A, A wild-type spikelet comprising a single floret enclosed by lemma and palea, a pair of rudimentary glumes (indicated by pink arrow heads) and a pair of empty glumes (indicated by white arrow heads). B to I, Individual spikelets from plants over-expressing miR172b. All of these spikelets do not have empty glumes but have multiple bract-like structures (top pair indicated by a pair of pink arrow heads). B, Fertile spikelet with a pair of lemma and palea (indicated by red *, same for C to G). C, Sterile spikelet with two pairs of lemma and palea. D, Sterile spikelet with multiple pairs of lemma and palea. The boxed part still contains multiple layers of lemma and palea. E, Sterile spikelet with a pair of twisted lemma and palea. F, Sterile spikelet with a normal-looking lemma or palea and a degenerated lemma or palea (indicated by blue *, same for H). G, Sterile spikelet with elongated lemma or palea. H, Sterile spikelet with multiple layers of degenerated lemma or palea. I, Sterile spikelet without floret. J, Scanning electron micrograph (SEM) of wild-type spikelet to show the surface features of empty glumes and rudimentary glumes. K, SEM of the bract-like structures of a spikelet from a miR172b over-expression plant. Green arrows in J and K indicate trichomes. L, Part of a wild-type panicle with normal spikelets. M, Part of a panicle from a miR172b over-expression plant with a strongly altered spikelet phenotype. N to P, Part of panicles from a miR172b over-expression plant with a moderately altered spikelet phenotype. White arrows in L to P indicate individual spikelets. Spikelets indicated by pink arrows in O and P represent spikelets with degenerated lemma or palea. Q, Wild-type mature seed. R to T, Mature seeds from plants over-expressing miR172b showing naked single grain (R and S) or double grains (T). EG: empty glume. LE: lemma. PA: palea. RG: rudimentary glume. Bars in A to I, and Q to T are 1 mm. Bars in J and K are 100 μm.
Mentions: We generated transgenic plants expressing the stem-loop precursors of miR172a and miR172b, transcribed by the maize ubiquitin promoter. Elevated levels of miR172 were detected in these miR172 over-expression plants, particularly in plants transformed with pre-MIR172b (Figure 4). Transgenic plants over-expressing miR172b showed normal vegetative growth and heading time, but the inflorescence (panicle) of the transformed plants was smaller, producing the half number of primary branches of the untransformed wild-type (Table 1). A wild-type spikelet consists of a single floret and two subtending pairs of bract-like structures - a pair of rudimentary glumes and a pair of empty glumes (Figure 5A, J). Spikelets of plants over-expressing miR172b were generated from primary or secondary branches as in wild-type, but the majority of spikelets were abnormal and showed variable defects in floral organs. The common phenotypes of the mutated spikelets were that more than two, and in extreme cases as many as 20, bract-like structures were generated before transition to floral development (Figure 5B to 5H); in some cases no obvious floral organs were produced (Figure 5I). The majority of these spikelets lacked a pair of empty glumes (compare Figure 5A with Figure 5B to 5H; Table 2). Scanning electron microscopy (SEM) showed that the lower part of the rudimentary glumes in the wild-type plant had round projections and small trichomes (Figure 5J). In the miR172b over-expression plants, similar round projections coated the surface of the bract-like structures though fewer trichomes were seen (Figure 5K), suggesting that the additional bract-like structures in the miR172b over-expression lines have the same identity as rudimentary glumes. This result suggests that reproductive development of plants over-expressing miR172b was not affected until the formation of the spikelet meristem, but the transition from spikelet meristem to floral meristem was delayed, leading to the reiteration of bract-like structures.

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