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A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D - PLoS Genet. (2010)

Bottom Line: Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants.That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development.In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

ABSTRACT
Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

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Details of defects observed in target mimic expressing plants.(A) Smaller flowers in severe MIM319 lines. The most strongly affected flowers lacked petals and did not have fully developed anthers (left side); in milder lines, flowers had short sepals, narrow petals, but were fertile (middle). Two flowers from wild type Col-0 are shown on the right side of the panel. (B) Severe MIM159 and MIM319 lines were very small and compact, without any stem elongation. (C) Leaves of MIM164 plants (compared to a leaf from wild type Col-0, on the far left). (D, E) Developing fruits of MIM164 with ectopic growths emanating from valve margins (D), which can develop into pseudo-pistils in severe lines (E). (F) Anthers in MIM167 lines did not mature completely (top), resulting in reduced pollen production (compared to a wild type Col-0 flower, bottom). (G) Seeds of MIM167 plants often do not fill completely, and remained attached to the dried silique (compared to a silique of wild type Col-0, on the right). (H, I) MIM171A lines suffered from defects in the separation of sepals, which prevented emergence of the pistil (H), and caused the plants to be mostly sterile (I, on the left, compared to a wild-type Col-0 plant, on the right). Bars correspond to 1 cm in (A–C) and I, and to 0.1 cm in (D–H).
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pgen-1001031-g003: Details of defects observed in target mimic expressing plants.(A) Smaller flowers in severe MIM319 lines. The most strongly affected flowers lacked petals and did not have fully developed anthers (left side); in milder lines, flowers had short sepals, narrow petals, but were fertile (middle). Two flowers from wild type Col-0 are shown on the right side of the panel. (B) Severe MIM159 and MIM319 lines were very small and compact, without any stem elongation. (C) Leaves of MIM164 plants (compared to a leaf from wild type Col-0, on the far left). (D, E) Developing fruits of MIM164 with ectopic growths emanating from valve margins (D), which can develop into pseudo-pistils in severe lines (E). (F) Anthers in MIM167 lines did not mature completely (top), resulting in reduced pollen production (compared to a wild type Col-0 flower, bottom). (G) Seeds of MIM167 plants often do not fill completely, and remained attached to the dried silique (compared to a silique of wild type Col-0, on the right). (H, I) MIM171A lines suffered from defects in the separation of sepals, which prevented emergence of the pistil (H), and caused the plants to be mostly sterile (I, on the left, compared to a wild-type Col-0 plant, on the right). Bars correspond to 1 cm in (A–C) and I, and to 0.1 cm in (D–H).

Mentions: MIM159 plants had extensive pleiotropic defects, and similar phenotypes were observed in most MIM319 lines. These plants had reduced stature, with rounder, upward curled leaves (Figure 2), shorter stem internodes, and smaller flowers with short sepals, reduced petals and anthers that did not develop completely. More severe MIM319 lines were progressively smaller, had warped leaves and lacked well-developed petals (Figure 3A). Stem elongation was often completely suppressed (Figure 3B). Most plants had reduced fertility, and this phenotype was particularly severe in MIM319 plants, for which only a few viable seeds could be recovered after they were grown for prolonged periods at 16°C in long days. Both vegetative and floral phenotypes reminiscent of MIM159 defects have been reported for plants that express non-targetable forms of miR159 target genes [29], and in plants doubly mutant for miR159a and miR159b [26]. In particular, upward curled leaves have been observed in plant expressing non-targetable forms of MYB33, which can be targeted both by miR159 and miR319 [43]. Milder MIM319 lines showed different leaf defects, with leaves curled downward (Figure 2). This is consistent with what has been reported for plants that express non-targetable forms of TCP2 and TCP4, which are both exclusive miR319 targets [29], suggesting that target mimics can at least partially discriminate between these two miRNA families.


A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D - PLoS Genet. (2010)

Details of defects observed in target mimic expressing plants.(A) Smaller flowers in severe MIM319 lines. The most strongly affected flowers lacked petals and did not have fully developed anthers (left side); in milder lines, flowers had short sepals, narrow petals, but were fertile (middle). Two flowers from wild type Col-0 are shown on the right side of the panel. (B) Severe MIM159 and MIM319 lines were very small and compact, without any stem elongation. (C) Leaves of MIM164 plants (compared to a leaf from wild type Col-0, on the far left). (D, E) Developing fruits of MIM164 with ectopic growths emanating from valve margins (D), which can develop into pseudo-pistils in severe lines (E). (F) Anthers in MIM167 lines did not mature completely (top), resulting in reduced pollen production (compared to a wild type Col-0 flower, bottom). (G) Seeds of MIM167 plants often do not fill completely, and remained attached to the dried silique (compared to a silique of wild type Col-0, on the right). (H, I) MIM171A lines suffered from defects in the separation of sepals, which prevented emergence of the pistil (H), and caused the plants to be mostly sterile (I, on the left, compared to a wild-type Col-0 plant, on the right). Bars correspond to 1 cm in (A–C) and I, and to 0.1 cm in (D–H).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908682&req=5

pgen-1001031-g003: Details of defects observed in target mimic expressing plants.(A) Smaller flowers in severe MIM319 lines. The most strongly affected flowers lacked petals and did not have fully developed anthers (left side); in milder lines, flowers had short sepals, narrow petals, but were fertile (middle). Two flowers from wild type Col-0 are shown on the right side of the panel. (B) Severe MIM159 and MIM319 lines were very small and compact, without any stem elongation. (C) Leaves of MIM164 plants (compared to a leaf from wild type Col-0, on the far left). (D, E) Developing fruits of MIM164 with ectopic growths emanating from valve margins (D), which can develop into pseudo-pistils in severe lines (E). (F) Anthers in MIM167 lines did not mature completely (top), resulting in reduced pollen production (compared to a wild type Col-0 flower, bottom). (G) Seeds of MIM167 plants often do not fill completely, and remained attached to the dried silique (compared to a silique of wild type Col-0, on the right). (H, I) MIM171A lines suffered from defects in the separation of sepals, which prevented emergence of the pistil (H), and caused the plants to be mostly sterile (I, on the left, compared to a wild-type Col-0 plant, on the right). Bars correspond to 1 cm in (A–C) and I, and to 0.1 cm in (D–H).
Mentions: MIM159 plants had extensive pleiotropic defects, and similar phenotypes were observed in most MIM319 lines. These plants had reduced stature, with rounder, upward curled leaves (Figure 2), shorter stem internodes, and smaller flowers with short sepals, reduced petals and anthers that did not develop completely. More severe MIM319 lines were progressively smaller, had warped leaves and lacked well-developed petals (Figure 3A). Stem elongation was often completely suppressed (Figure 3B). Most plants had reduced fertility, and this phenotype was particularly severe in MIM319 plants, for which only a few viable seeds could be recovered after they were grown for prolonged periods at 16°C in long days. Both vegetative and floral phenotypes reminiscent of MIM159 defects have been reported for plants that express non-targetable forms of miR159 target genes [29], and in plants doubly mutant for miR159a and miR159b [26]. In particular, upward curled leaves have been observed in plant expressing non-targetable forms of MYB33, which can be targeted both by miR159 and miR319 [43]. Milder MIM319 lines showed different leaf defects, with leaves curled downward (Figure 2). This is consistent with what has been reported for plants that express non-targetable forms of TCP2 and TCP4, which are both exclusive miR319 targets [29], suggesting that target mimics can at least partially discriminate between these two miRNA families.

Bottom Line: Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants.That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development.In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

ABSTRACT
Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

Show MeSH
Related in: MedlinePlus