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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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Accumulation of miR166 and miR390 within the SAM is regulated at the level of biogenesis and/or stability.(A) Laser-microdissection of domains within the SAM: 5) the stem cell containing SAM tip; 6) the incipient primordium; 7) the region below the incipient leaf. (B) Expression analysis of mir166 family members by RT-PCR shows that only mir166a, -c, -f and –i precursor transcripts accumulate in or below the incipient leaf and likely contribute to the establishment of organ polarity. In addition, mir166a transcripts are expressed in the SAM tip. Whereas both mir390 precursors are expressed in the tip of the SAM and in the incipient leaf, only mir390a accumulates in the region below the incipient leaf. (C) In situ hybridization of a longitudinal section through a wild-type apex with a miR166 complementary LNA probe shows miR166 accumulates below the incipient leaf and on the abaxial side of the incipient and developing leaf primordia. Note the lack of detectable signal in the tip of the SAM (marked “M”). (D) Longitudinal section through a wild-type apex shows rld2 expression in the tip of the SAM, vasculature, and the adaxial side of leaf primordia. (E) Expression of tub6, kan1, kan2, and rld1 in the captured SAM regions is as previously reported [7],[23], illustrating the accuracy of laser-microdissection. (F) RT-PCR analysis for the core miRNA processing genes dcl1, se1a and se1b in the SAM tip.
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pgen-1000320-g002: Accumulation of miR166 and miR390 within the SAM is regulated at the level of biogenesis and/or stability.(A) Laser-microdissection of domains within the SAM: 5) the stem cell containing SAM tip; 6) the incipient primordium; 7) the region below the incipient leaf. (B) Expression analysis of mir166 family members by RT-PCR shows that only mir166a, -c, -f and –i precursor transcripts accumulate in or below the incipient leaf and likely contribute to the establishment of organ polarity. In addition, mir166a transcripts are expressed in the SAM tip. Whereas both mir390 precursors are expressed in the tip of the SAM and in the incipient leaf, only mir390a accumulates in the region below the incipient leaf. (C) In situ hybridization of a longitudinal section through a wild-type apex with a miR166 complementary LNA probe shows miR166 accumulates below the incipient leaf and on the abaxial side of the incipient and developing leaf primordia. Note the lack of detectable signal in the tip of the SAM (marked “M”). (D) Longitudinal section through a wild-type apex shows rld2 expression in the tip of the SAM, vasculature, and the adaxial side of leaf primordia. (E) Expression of tub6, kan1, kan2, and rld1 in the captured SAM regions is as previously reported [7],[23], illustrating the accuracy of laser-microdissection. (F) RT-PCR analysis for the core miRNA processing genes dcl1, se1a and se1b in the SAM tip.

Mentions: To determine which mir166 genes act in the SAM to establish the abaxial-graded pattern of miR166 in the incipient leaf [3],[7] and therefore contribute to the specification of leaf polarity, we next microdissected precisely defined domains within the SAM. Expression of mir166a through mir166i was evaluated in cells captured from below the incipient leaf, the incipient primordium, as well as the tip of the SAM in which mature miR166 does not accumulate (Figure 2A). The expression profiles of the control genes kan1, kan2 and rld1 in these domains recapitulate their described expression patterns and thus verify the purity of the LM samples (Figure 2E). Transcripts of only a subset of mir166 family members are detected in the SAM. mir166a, -f and -i are expressed both in and below the incipient primordium, whereas mir166c is expressed at detectable levels exclusively below the initiating leaf (Figure 2B). This data further supports the existence of functional diversification within the mir166 gene family and shows that a subset of mir166 precursor genes contributes to the abaxial accumulation of mature miR166 that is required for establishing abaxial fate.


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)

Accumulation of miR166 and miR390 within the SAM is regulated at the level of biogenesis and/or stability.(A) Laser-microdissection of domains within the SAM: 5) the stem cell containing SAM tip; 6) the incipient primordium; 7) the region below the incipient leaf. (B) Expression analysis of mir166 family members by RT-PCR shows that only mir166a, -c, -f and –i precursor transcripts accumulate in or below the incipient leaf and likely contribute to the establishment of organ polarity. In addition, mir166a transcripts are expressed in the SAM tip. Whereas both mir390 precursors are expressed in the tip of the SAM and in the incipient leaf, only mir390a accumulates in the region below the incipient leaf. (C) In situ hybridization of a longitudinal section through a wild-type apex with a miR166 complementary LNA probe shows miR166 accumulates below the incipient leaf and on the abaxial side of the incipient and developing leaf primordia. Note the lack of detectable signal in the tip of the SAM (marked “M”). (D) Longitudinal section through a wild-type apex shows rld2 expression in the tip of the SAM, vasculature, and the adaxial side of leaf primordia. (E) Expression of tub6, kan1, kan2, and rld1 in the captured SAM regions is as previously reported [7],[23], illustrating the accuracy of laser-microdissection. (F) RT-PCR analysis for the core miRNA processing genes dcl1, se1a and se1b in the SAM tip.
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Related In: Results  -  Collection

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pgen-1000320-g002: Accumulation of miR166 and miR390 within the SAM is regulated at the level of biogenesis and/or stability.(A) Laser-microdissection of domains within the SAM: 5) the stem cell containing SAM tip; 6) the incipient primordium; 7) the region below the incipient leaf. (B) Expression analysis of mir166 family members by RT-PCR shows that only mir166a, -c, -f and –i precursor transcripts accumulate in or below the incipient leaf and likely contribute to the establishment of organ polarity. In addition, mir166a transcripts are expressed in the SAM tip. Whereas both mir390 precursors are expressed in the tip of the SAM and in the incipient leaf, only mir390a accumulates in the region below the incipient leaf. (C) In situ hybridization of a longitudinal section through a wild-type apex with a miR166 complementary LNA probe shows miR166 accumulates below the incipient leaf and on the abaxial side of the incipient and developing leaf primordia. Note the lack of detectable signal in the tip of the SAM (marked “M”). (D) Longitudinal section through a wild-type apex shows rld2 expression in the tip of the SAM, vasculature, and the adaxial side of leaf primordia. (E) Expression of tub6, kan1, kan2, and rld1 in the captured SAM regions is as previously reported [7],[23], illustrating the accuracy of laser-microdissection. (F) RT-PCR analysis for the core miRNA processing genes dcl1, se1a and se1b in the SAM tip.
Mentions: To determine which mir166 genes act in the SAM to establish the abaxial-graded pattern of miR166 in the incipient leaf [3],[7] and therefore contribute to the specification of leaf polarity, we next microdissected precisely defined domains within the SAM. Expression of mir166a through mir166i was evaluated in cells captured from below the incipient leaf, the incipient primordium, as well as the tip of the SAM in which mature miR166 does not accumulate (Figure 2A). The expression profiles of the control genes kan1, kan2 and rld1 in these domains recapitulate their described expression patterns and thus verify the purity of the LM samples (Figure 2E). Transcripts of only a subset of mir166 family members are detected in the SAM. mir166a, -f and -i are expressed both in and below the incipient primordium, whereas mir166c is expressed at detectable levels exclusively below the initiating leaf (Figure 2B). This data further supports the existence of functional diversification within the mir166 gene family and shows that a subset of mir166 precursor genes contributes to the abaxial accumulation of mature miR166 that is required for establishing abaxial fate.

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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