Article Figures & Data
Figures
- Figure 1.
Proposed Model for the Role of Small Regulatory RNAs in Dorsiventral Patterning.
Model modified from Kidner and Timmermans (2007). Details provided in text. AGO7/RGD2, ARGONAUTE7/RAGGED SEEDLING2; miR390, microRNA 390; SGS3/LBL1, SUPPRESSOR-OF-GENE SILENCING3/LEAFBLADELESS1; RDR6, RNA-DEPENDANT RNA POLYMERASE6; DCL4, DICER-LIKE4; AGO1, ARGONAUTE1; ARF3a, AUXIN RESPONSE FACTOR3a; miR166, microRNA 166; HD-ZIPIII, family of adaxial identity factors.
- Figure 2.
Positional Cloning and Characterization of rgd2 Alleles.
(A) Positional cloning of rgd2. rgd2-R was mapped to bin 1.04 using simple sequence repeat markers (names) and fine mapped to an interval flanked by CAP76M21 and IDP1473. Candidate gene analysis revealed an ago7-like locus, GRMZM2G365589, on maize BAC c0230J20. Gray blocks indicate BAC clones. cM, centimorgan.
(B) Gene structure of rgd2 wild-type and mutant alleles. The rgd2 open reading frame consists of three exons (black boxes) and shows a high degree of similarity to ago7. Shaded boxes show the approximate positions of the predicted PAZ and PIWI protein domains. Solid gray box represents 3′ untranslated region. Inverted triangles mark the locations of transposon insertions in the rgd2-R and rgd2-Ds1 alleles. Point mutations in the EMS-induced alleles, rgd2-e1 and rgd2-e2, are denoted. Images of representative phenotypes of rgd2 mutant alleles are placed above each corresponding mutation. Bars = 1 cm.
(C) RT-PCR analysis of rgd2 transcripts. Primers F1 and R1, which flank the first intron, were used in rgd2 transcript analyses of seedlings from rgd2-R mutants, wild-type siblings, and heterozygous siblings. No transcripts were detected in rgd2-R mutants after 42 amplification cycles. RT-PCR using tub6 primers served as the control. Three biological replicates comprising one seedling per replicate were performed.
[See online article for color version of this figure.]
- Figure 3.
In Situ Hybridization Analysis of rgd2 and miR390 Accumulation in rgd2 Mutant and Wild-Type Sibling Apices.
(A) and (B) Accumulation pattern of rgd2 in the SAM (asterisks) and leaf primordia (P1 and P2).
(A) Transverse wild-type shoot apex.
(B) Longitudinal wild-type shoot apex.
(C) Control in situ hybridization of wild-type shoot apex using a sense rgd2 hybridization probe.
(D) to (G) Accumulation pattern of miR390.
(D) Transverse wild-type shoot apex.
(E) Longitudinal wild-type shoot apex.
(F) Longitudinal rgd2-R shoot apices show ectopic miR390 expression throughout the SAM crown and leaf primordia.
(G) Longitudinal rgd2-e2 shoot apex.
(H) An LNA probe for murine miR124e was used as a sense control for miR390 accumulation.
Bars = 100 μm; arrows indicate areas of transcript accumulation.
[See online article for color version of this figure.]
- Figure 4.
Accumulation Analyses of Transcripts Involved in the ta-siARF Pathway.
(A) Small RNA gel blot hybridization of miR390, miR166, and ta-siARF in wild-type and rgd2-R shoot apices. All genotypes are introgressed into the Mo17 genetic background.
(B) qRT-PCR analysis of arf3a and tas3a in laser-microdissected leaf primordia and SAMs from wild-type (WT) and rgd2-R mutants. Accumulation was normalized to ubq. Three biological replicates were performed per experiment. Error bars denote 1 se.
(C) qRT-PCR analysis of miR166 precursors in laser-microdissected SAMs from wild-type and rgd2-R mutants. An ago1 putative paralogue (1GenBank accession number AY110984) was analyzed in wild-type, rgd2-R mutant, and lbl1-ref mutant 14-d-old seedlings. Accumulation was normalized to tub6. Each experiment used three biological replicates. Error bars denote 1 se.
- Figure 5.
In Situ Hybridization Analysis of tas3a and arf3a in the Wild Type and in rgd2-R and lbl1-rgd1 Mutants.
(A) Accumulation of tas3a in a wild-type apex transverse section using a tas3a antisense hybridization probe. (B) to (D) Pattern of transcript accumulation obtained using the ta-siARF LNA hybridization probe. (B) Transverse section of a wild-type apex. Note that the pattern of transcript accumulation detected with the ta-siARF LNA probe is equivalent to that observed when using the tas3a probe (A). (C) Longitudinal section of a wild-type SAM (asterisk) and leaf primordia (P1 and P2) (D) Longitudinal section of a rgd2-R mutant apex shows ectopic transcript accumulation in the SAM and the leaf primordia. (E) Control in situ hybridization of wild-type shoot apex using a sense tas3a hybridization probe. (F) to (H) Accumulation pattern of arf3a transcripts. (F) Longitudinal section of a wild-type apex reveals arf3a transcript accumulation in abaxial domains of leaf primordia. (G) and (H) Longitudinal sections of rg2-R (G) and lbl1-rgd1 (H) mutant apices; arf3a transcript accumulation remains abaxialized in both. (I) Control in situ hybridization of wild-type shoot apex using a sense arf3a hybridization probe. (J) The LNA ta-siARF in situ hybridization probe is predicted to hybridize to the mature ta-siARF as well as to two locations in its abundant precursor, the tas3a transcript. Bars = 100 μm. Arrows point to areas of accumulation.
[See online article for color version of this figure.]
- Figure 6.
In Situ Hybridization Analysis of miR166 in the Wild Type and rgd2-R Mutants.
(A) and (B) Accumulation of miR166 in shoot apices. Bars = 50 μm.
(A) Longitudinal section of a wild-type apex shows miR166 accumulation in abaxial regions of leaf primordia (indicated by arrows). Asterisk denotes SAM.
(B) Longitudinal section of an rgd2-R mutant apex shows ectopic accumulation of miR166 in adaxial regions of leaf primordia and the crown of the meristem (arrows).
[See online article for color version of this figure.]
- Figure 7.
Revised Model for the Role of Small Regulatory RNAs in Dorsiventral Patterning.
rgd2 is the maize homolog of Arabidopsis ago7 and is required to regulate miR390 accumulation and localization in the maize shoot apex. RGD2 and LBL1/SGS3 are both required to regulate miR166 accumulation levels. Like LBL1, RGD2 is required to properly localize miR166, suggesting that miR166 polarization requires ta-siARF function. Although rgd2 mutants overaccumulate miR166 and arf3a, miR166 is properly localized, resulting in no net loss of dorsiventral patterning in rgd2 mutants. However, in lbl1 mutants, the ectopic accumulation of miR166 combined with the hyperaccumulation of ago1 may account for the abaxialized mutant leaf phenotype. Abbreviations are as described in Figure 1.
[See online article for color version of this figure.]
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