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Regulation of small RNA accumulation in the maize shoot apex.

Nogueira FT, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MC - PLoS Genet. (2009)

Bottom Line: Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity.Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well.Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

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mir166 family members exhibit distinct overlapping expression profiles in the maize shoot apex.(A) Longitudinal sections through a maize apex diagramming regions captured by laser-microdissection (left panel): 1) P2–P3 leaf primordia; 2) the incipient (P0) and P1 leaf primordia plus the base of the SAM; 3) P4–P6 leaf primordia; 4) stem tissue. The right panel shows a longitudinal section of an apex after capturing cells from the P2 and P3 leaf primordia. Note the precision with which cells are captured. (B) Transcript accumulation of tub6, rld1, rs1 and kan1 in these regions is as previously reported [7], [22]–[23], illustrating the accuracy of laser-microdissection. (C) RT-PCR analysis of mir166a to mir166e shows that these mir166 family members exhibit distinct but overlapping expression profiles within the vegetative apex. -RT controls are also shown.
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pgen-1000320-g001: mir166 family members exhibit distinct overlapping expression profiles in the maize shoot apex.(A) Longitudinal sections through a maize apex diagramming regions captured by laser-microdissection (left panel): 1) P2–P3 leaf primordia; 2) the incipient (P0) and P1 leaf primordia plus the base of the SAM; 3) P4–P6 leaf primordia; 4) stem tissue. The right panel shows a longitudinal section of an apex after capturing cells from the P2 and P3 leaf primordia. Note the precision with which cells are captured. (B) Transcript accumulation of tub6, rld1, rs1 and kan1 in these regions is as previously reported [7], [22]–[23], illustrating the accuracy of laser-microdissection. (C) RT-PCR analysis of mir166a to mir166e shows that these mir166 family members exhibit distinct but overlapping expression profiles within the vegetative apex. -RT controls are also shown.

Mentions: As an initial analysis, we investigated the expression of a subset of mir166 genes in relatively broad domains of the maize apex (Figure 1). Cells were captured from regions in which miR166 accumulates [7], including: 1) the P2 and P3 developing leaf primordia; 2) the incipient leaf (P0) where polarity is established, the P1, and the region of tissue just below the SAM; 3) more developed P4-P6 primordia; and 4) stem tissue, which contains extensive vasculature (Figure 1A). The expression profile of selected control genes in the microdissected domains was tested to determine the accuracy of LM. Consistent with their reported in situ hybridization expression patterns, the miR166 target rolled leaf1 (rld1) is expressed in all tissue samples tested [7], rough sheath1 (rs1) transcripts are limited to those domains that include the subtending regions of leaves [22], and similar to kanadi2 (kan2), the expression of kan1 demarks cells in the developing young leaf primordia [23] (Figure 1B).


Regulation of small RNA accumulation in the maize shoot apex.

Nogueira FT, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MC - PLoS Genet. (2009)

mir166 family members exhibit distinct overlapping expression profiles in the maize shoot apex.(A) Longitudinal sections through a maize apex diagramming regions captured by laser-microdissection (left panel): 1) P2–P3 leaf primordia; 2) the incipient (P0) and P1 leaf primordia plus the base of the SAM; 3) P4–P6 leaf primordia; 4) stem tissue. The right panel shows a longitudinal section of an apex after capturing cells from the P2 and P3 leaf primordia. Note the precision with which cells are captured. (B) Transcript accumulation of tub6, rld1, rs1 and kan1 in these regions is as previously reported [7], [22]–[23], illustrating the accuracy of laser-microdissection. (C) RT-PCR analysis of mir166a to mir166e shows that these mir166 family members exhibit distinct but overlapping expression profiles within the vegetative apex. -RT controls are also shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602737&req=5

pgen-1000320-g001: mir166 family members exhibit distinct overlapping expression profiles in the maize shoot apex.(A) Longitudinal sections through a maize apex diagramming regions captured by laser-microdissection (left panel): 1) P2–P3 leaf primordia; 2) the incipient (P0) and P1 leaf primordia plus the base of the SAM; 3) P4–P6 leaf primordia; 4) stem tissue. The right panel shows a longitudinal section of an apex after capturing cells from the P2 and P3 leaf primordia. Note the precision with which cells are captured. (B) Transcript accumulation of tub6, rld1, rs1 and kan1 in these regions is as previously reported [7], [22]–[23], illustrating the accuracy of laser-microdissection. (C) RT-PCR analysis of mir166a to mir166e shows that these mir166 family members exhibit distinct but overlapping expression profiles within the vegetative apex. -RT controls are also shown.
Mentions: As an initial analysis, we investigated the expression of a subset of mir166 genes in relatively broad domains of the maize apex (Figure 1). Cells were captured from regions in which miR166 accumulates [7], including: 1) the P2 and P3 developing leaf primordia; 2) the incipient leaf (P0) where polarity is established, the P1, and the region of tissue just below the SAM; 3) more developed P4-P6 primordia; and 4) stem tissue, which contains extensive vasculature (Figure 1A). The expression profile of selected control genes in the microdissected domains was tested to determine the accuracy of LM. Consistent with their reported in situ hybridization expression patterns, the miR166 target rolled leaf1 (rld1) is expressed in all tissue samples tested [7], rough sheath1 (rs1) transcripts are limited to those domains that include the subtending regions of leaves [22], and similar to kanadi2 (kan2), the expression of kan1 demarks cells in the developing young leaf primordia [23] (Figure 1B).

Bottom Line: Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity.Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well.Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

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