Grant-Downton Robert , Hafidh Said , Twell David , Dickinson Hugh G.

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Small RNA Pathways Are Present and Functional in the Angiosperm Male Gametophyte Grant-Downton Robert , Hafidh Said , Twell David , Dickinson Hugh G. Molecular Plant Volume 2, Issue 3, Pages 500-512 (May 2009) DOI: 10.1093/mp/ssp003 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Expression Profiles of sRNA Pathway-Related Genes at Four Stages of Pollen Development. (A) Plot of transcriptomic profiles for 39 sRNA pathway-related genes (see Supplemental Table 1) based on normalized ATH1 Affymetrix chip data (Dupl'áková et al., 2007). (B–F) RT–PCR analysis of sRNA pathway genes during pollen development. (B) AGO family members, (C) DCL1-4, (D) HASTY, SERRATE, HEN1, (E) six members of the RDR family, (F) HISTONE H3 (AT4G40040) was used as a control. UNM, unicellular microspores; BCP, bicellular pollen; TCP, tricellular pollen; MPG, mature pollen grains. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Mature miRNA Amplifications from Endpoint RT–PCRs from cDNAs of Small RNAs below 300 nt. V, vegetative control (aerial sporophyte); P, mature pollen; L, DNA ladder; C, control (cDNA only, no miRNA). Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Pri-miRNA Amplifications from cDNA of Total RNA from Vegetative Control (V, Aerial Sporophyte) and Mature Pollen (P). Identity of miRNA shown on lanes; L, DNA ladder. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Pre-miRNA Amplifications from cDNA of Total RNA from Vegetative Control (Aerial Sporophyte, V) and Mature Pollen (P). NC, negative control (cDNA only, no miRNA amplification); L, DNA ladder. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Abundance of ARF17 and TOE2 Transcripts (RT–PCR) in Mature Pollen (P) and Sporophyte (V). Two sites on the transcripts were amplified, with primers flanking the cleavage sites (CS) and close to the 3’ terminus (3’). RNA-only controls gave no bands (data not shown). L, DNA ladder. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 5′ RACE Showing Products from AGO1, DUO1, and ARF16 Nested Amplifications in Mature Pollen and Vegetative Control. P, mature pollen; PC, mature pollen control (gene-specific reverse nested primer only); V, vegetative plant (sporophyte); VC, vegetative plant (sporophyte); NC, negative control (gene-specific reverse nested primer only); L, DNA ladder. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 5′ RACE Showing Products from ARF17 and TOE2 Nested Amplifications in Mature Pollen and Vegetative Control. P, mature pollen; PC, mature pollen control (gene-specific reverse nested primer only); V, vegetative plant (sporophyte); VC, vegetative plant (sporophyte); NC, negative control (gene-specific reverse nested primer only); L, DNA ladder. Molecular Plant 2009 2, 500-512DOI: (10.1093/mp/ssp003) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions