Supporting Information for

A role for the shoot apical meristem in the specification of juvenile leaf identity in

Arabidopsis

Jim P. Fouracre and R. Scott Poethig

Correspondence to spoethig@sas.upenn.edu

This PDF includes:

Materials and Methods

Supplementary Figures 1-6

Supplementary figure legends

Supplementary Table 1

SI references

Materials and Methods

Plant material and growth conditions

Col was used as the genetic background for all stocks unless specified. wus-5

(SAIL_150_G06) and clv3-10 (CS68823) were obtained from the Arabidopsis Biological

Resource Center (Ohio State University). The following lines have been previously

described: clv1-4 (1); 35S::MIM172 (2); SPL9::SPL9-GUS, SPL9::rSPL9-GUS,

SPL13::SPL13-GUS, SPL13::rSPL13-GUS and spl2/9/10/11/13/15 (3); mir156a mir156c

mir157a mir157c (4); stm-1 introgressed into Col (5); psd-13 (6) and LFY::GUS (7).

www.pnas.org/cgi/doi/10.1073/pnas.1817853116

Seeds were sown on fertilized Farfard #2 soil (Farfard) and kept at 4oC for 3 days prior

to transfer to a growth chamber, with the transfer day counted as day 0 for plant age (0

DAP). Plants were grown at 22oC under a mix of both white (USHIO F32T8/741) and

red-enriched (Interlectric F32/T8/WS Gro-Lite) fluorescent bulbs in either long day (16

hrs. light/8 hrs. dark; 95 μmol m-2 s-1) or short day (10 hrs light/14 hrs dark; 180 μmol m-

2 s-1) conditions.

Generation of transgenic plants

Arabidopsis genomic DNA was used as a template for the cloning of all promoter

sequences and the MIR156A locus. Promoter sequences were defined as the following

regions upstream of the translational start site: WUS – 1.7kb, STM – 5.75kb, ANT – 6kb

and FD – 3kb. For MIR156A, 1.8kb extending 500bp upstream and 1.3kb downstream

of the mature miRNA was used. The target mimic MIMIC156 was recloned from the

original published vector (2). Type II restriction sites were cloned out of all sequences

and the Golden Gate cloning strategy utilized to generate binary vectors using the

MoClo cloning toolbox (8) provided by Addgene (www.addgene.org). pAGM4723 was

used as the final binary vector in all cases and was assembled, along with intermediary

Golden Gate modules, using BsaI or BpiI (NEB) and T4 DNA Ligase (NEB). The MoClo

pAtuOcs sequence was used as a transcriptional terminator for all MIR156A and

MIMIC156 constructs, the GUS sequence for promoter::GUS fusion constructs and the

pFAST-R selection cassette as a selectable marker. The GV3101 strain of

Agrobacterium was used for plant transformation by floral dipping (9). Single insertion

events were identified by seed fluorescent strength in the T1 generation and a 3:1 ratio

of fluorescence in the T2. The sequences of all cloning primers are included in

Supplementary Table 1.

GUS staining and histology

Plants were fixed in 90% acetone on ice for 10 minutes and washed with GUS staining

buffer (5mM potassium ferricyanide and 5mM ferrocyanide in 0.1M PO4 buffer) and

stained for between 8 hrs and overnight (depending on transgene strength) at 37oC in

2mM X-Gluc GUS staining buffer. A MUG assay was carried out to determine GUS

activity quantitatively as previously described (4). For histological observations

individuals were fixed in FAA (3.7% formaldehyde), dehydrated in an ethanol series and

cleared using Histo-Clear (National Diagnostics). Following embedding in Paraplast

Plus (Sigma-Aldrich) 8μM sections were produced using an HM 355 microtome

(Microm) and visualized using an Olympus BX51 microscope with a DP71 camera

attachment (Olympus). ImageJ software (www.imagej.net) was used to measure

meristem sizes.

RNA expression analysis

Tissue (either shoot apices with leaf primordia £1mm, isolated leaf primordia 1-2mm in

size or cotyledons – as specified in the text) were ground in liquid nitrogen and total

RNA extracted using Trizol (Invitrogen) as per the manufacturer’s instructions. RNA was

DNAse treated with RQ1 (Promega) and 1μg of RNA was used for reverse transcription

using Superscript III (Invitrogen). Gene specific RT primers were used to amplify

miR156, miR157 and SnoR101 (10) and a polyT primer for mRNA amplification. Three-

step qPCR of cDNA was carried out using either Platinum Taq (Invitrogen) and

EvaGreen (Biotium) or SYBR-Green Master Mix (Bimake). qPCR reactions were run in

triplicate and an average taken to produce a single biological replicate. The data

presented represents the average of distinct biological replicates (the number of which

are specified in the text).

Statistical analysis

A two-tailed Student’s t-test was used to carry out pairwise comparisons between

different genotypes. For comparison of multiple samples, to decrease the chance of

false positives, a one-way ANOVA followed by either two-tailed Dunnett’s or Tukey tests

were used to compare multiple transgenic lines to either a single sample (i.e. ‘many-to-

one’; Dunnett’s test) or for multi-way comparisons (i.e. ‘many-to-many’; Tukey test).

Statistical analyses were carried out in R (r-project.org) and Excel (Microsoft), sample

sizes and P values are specified in the text.

Fig. S1. The wus leaf phenotype is not explained by a delay in leaf initiation or

emergence. (A) Examples of LFY::GUS expression in different meristem defective

mutants. Scale bar = 1mm. (B, C) Silhouetes of fully expanded leaves 1 and 2 of WT (B)

and wus (C) plants. DAP indicates the day at which the primordia of these leaves visibly

emerged.

Fig. S2. Expression of miR156 in cotyledons is unaffected in wus plants. Cotyledons

were harvested at 6 DAP in LD conditions. miR156 levels were quantified by qPCR,

normalized to snoR101 as an internal control gene and expressed as a ratio of

expression in WT plants. Each data point represents a biological replicate and is the

average of three technical replicates. Black bars represent the mean and grey bars the

standard error of the mean.

Fig. S3. Molecular and phenotypic characterisation of miR156-manipulation lines. (A)

GUS staining of promoter::GUS fusions for promoters used to drive MIR156A and

MIM156 expression. Sections show plants at 2 weeks in SD conditions. Scale bars =

100um. (B) Gene expression analyses of miR156 and SPL9 in 7 DAP seedlings in LD

conditions for the lines shown in Fig. 4. Relative levels were quantified by qPCR,

normalized to snoR101 (for miR156) or ACT2 (for SPL9) as internal control genes and

expressed as a ratio of expression in WT plants. Each data point represents a biological

replicate and is the average of three technical replicates. Black bars represent the mean

and grey bars the standard error of the mean. (C) All rosette leaves (except for

ANT::MIR156A for which the first 12 are shown) for an individual plant are displayed in

order of emergence, with the youngest leaf on the left, plants were grown in LD

conditions.

Fig. S4. Persistent expression of miR156 in the shoot apex delays VPC and flowering

relative to WT plants in non-inductive conditions. qm plants are quadruple mutant for

mir156ac mir157ac. Two independent T3 lines are shown for FD::MIR156A; qm.

Significantly distinct groups were determined by one-way ANOVA with post hoc Tukey

multiple comparison test (letters indicate statistically distinct groups; P < 0.05; sample

sizes are indicated on the graph). Boxes display the interquartile range (IQR) (boxes),

median (lines) and values beyond 1.5*IQR (whiskers); mean values are marked by u.

Plants were grown in SD conditions.

Fig. S5. SAM-specific expression of miR156 is able to decrease SPL accumulation in

young leaf primordia. GUS-stained wax sections of plants that are homozygous quadruple

mutant (qm) for mir156ac mir157ac and hemizygous for a SPL9::SPL9-GUS reporter

construct. (A) qm control. (B) plants hemizygous for WUS::MIR156A. (C) plants

hemizygous for STM::MIR156A. Black arrowheads show reduced accumulation of SPL9-

GUS in young leaf primordia. Black scale bar = 100µm.

Supplementary Table 1: Primers used in this study.

Primer name Sequence Golden Gate cloning primers proWUS_GG_L-1_F1 ttggtctcaacatggagCCAATATAATCGACTAAAGTTAAAAA

proWUS_GG_L-1_R1 ttggtctcaGAAAACAGGTGTACTTATGAACATG

proWUS_GG_L-1_F2 ttggtctcaTTTCACACTCGTTTCACACATTGTAATTG

proWUS_GG_L-1_R2 ttggtctcaacaacattGTGTGTTTGATTCGACTTTTGTTCAC

proSTM_GG_L-1_F1 ttggtctcaacatggagATTATAACCCAACCTAGCTATCTTCAAC

proSTM_GG_L-1_R1 ttggtctcaAAGGCTCAATACCATGGAAATGAGC

proSTM_GG_L-1_F2 ttggtctcaCCTTCTATATGCGAGAGAATCAGATTC

proSTM_GG_L-1_R2 ttggtctcaGTCCTCTCTCTCTAAAGCCCTAATG

proSTM_GG_L-1_F3 ttggtctcaGGACATTCGGACTGTCCCCACTTG

proSTM_GG_L-1_R3 ttggtctcaCCTCTCATTTAAGAACCAAATAGTC

proSTM_GG_L-1_F4 ttggtctcaGAGGCCCCCGAAAAAATCTTT

proSTM_GG_L-1_R4 ttggtctcaAGATCAGGAAGATAAATCTAAGG

proSTM_GG_L-1_F5 ttggtctcaATCTCTCTTCTGCTGCTTCCCTCTC

proSTM_GG_L-1_R5 ttggtctcaTGGCCTCCCGGGATTTATGCTTC

proSTM_GG_L-1_F6 ttggtctcaGCCACTTTTGCATTTTCAAATAATT

proSTM_GG_L-1_R6 ttggtctcaacaacattCTTCTCTTTCTCTCACTAGTATTA

proANT_GG_L-1_F1 ttggtctcaacatggagTTTTGGAGTTTTCTTCATTATATG proANT_GG_L-1_R1b ttggtctcaTCAAAAAGTCCAAAACAATCC

proANT_GG_L-1_F2b ttggtctcaTTGATCTTCGAAGTTTCAAGCTG

proANT_GG_L-1_R2b ttggtctcaTTTCGTTGAAAAGAAACTCTCTGTAATC

proANT_GG_L-1_F3b ttggtctcaGAAAACAAAAAAAAGAAAAGG

proANT_GG_L-1_R3 ttggtctcaTCTAATCTCGATTGTCATTAGAC

proANT_GG_L-1_F4 ttggtctcaTAGACCTGATATAAAACAAAAACAGATAC

proANT_GG_L-1_R4c ttggtctcaACTTAACAGAGCATGTCCTCTACTTTTC

proANT_GG_L-1_F5b ttggtctcaAAGTGGTCGCTGTTTTCACTC

proANT_GG_L-1_R5 ttggtctcaGAACACACTCAACATATTTAAGTTTG

proANT_GG_L-1_F6 ttggtctcaGTTCAGTGCTCACTGTTCAGG

proANT_GG_L-1_R6 ttggtctcaGACAACTCTTTGGCTTCATGC

proANT_GG_L-1_F7 ttggtctcaTGTCTCTGTCCTAAAGATATCTACAGC

proANT_GG_L-1_R7 ttggtctcaAGATGAGTTACAATACAACTGATGACAG

proANT_GG_L-1_F8 ttggtctcaATCTCTTAGCCATATAGTTCTAAG

proANT_GG_L-1_R8 ttggtctcaacaacattGGTTTCTTTTTTTGGTTTCTGC

proFD_GG_L-1_F1 ttggtctcaacatggagTAGTTATCCAAGGCCCTCTCTACTTG

proFD_GG_L-1_R1 ttggtctcaGAGGCGTAAAAGGGTTTAAGATAGAC

proFD_GG_L-1_F2 ttggtctcaCCTCTAAATATATAGAGATGTAATTAGTATT

proFD_GG_L-1_R2 ttggtctcaCACATCTCAGGGGATTGG

proFD_GG_L-1_F3 ttggtctcaTGTGACATGTCACACTCCTTTC

proFD_GG_L-1_R3 ttggtctcaAGATGAATACTAAACTAGTTAATAATTAAG

proFD_GG_L-1_F4 ttggtctcaATCTCTAATCTTCAAAACAATCAAC

proFD_GG_L-1_R4 ttggtctcaTGGATCTCCAACTCTGAAC

proFD_GG_L-1_F5 ttggtctcaTCCACATAAATTGATTTCCCTATC

proFD_GG_L-1_R5 ttggtctcaTGTATTCAACGTCAGAGTTATTATAAAG

proFD_GG_L-1_F6 ttggtctcaTACATCAACAAACACCTTTAATTAC

proFD_GG_L-1_R6 ttggtctcaacaacattTGGAAAAGAGAACAGAAGTGAACC

cdsMIR156A_GG_L-1_F1

ttggtctcaacataatgGTTCACTCTCAAATCTCAAGTTCATTGCCATTTTTAGTT

cdsMIR156A_GG_L-1_R1 ttggtctcaGACTCTTTAGAAGATCAAATCTAGGGTTTTTG

cdsMIR156A_GG_L-1_F2 ttggtctcaAGTCTCAAATGGAATCTCTTCTC

cdsMIR156A_GG_L-1_R2 ttggtctcaGAGGAGACAAAGAATCAAAGAGAG

cdsMIR156A_GG_L-1_F3 ttggtctcaCCTCCAGTTAAAACTCAGATCTAAC

cdsMIR156A_GG_L-1_R3 ttggtctcaGAAAACAGGCCAAAGAGATC

cdsMIR156A_GG_L-1_F4 ttggtctcaTTTCGTTCTCTATGTCTCAATCTCTCTC

cdsMIR156A_GG_L-1_R4 ttggtctcaAGGCCTCCTCCCGCATG

cdsMIR156A_GG_L-1_F5 ttggtctcaGCCTTTAGCCTTTAATCATTCATTATATTTTAAG

cdsMIR156A_GG_L-1_R5 ttggtctcaGGAGGACATGACACATCAACTTG

cdsMIR156A_GG_L-1_F6 ttggtctcaCTCCCACTTTTGTACTGTTAATACTG

cdsMIR156A_GG_L-1_R6 ttggtctcaTGTTTTCCAAATTTCCCAATC

cdsMIR156A_GG_L-1_F7 ttggtctcaAACATGGAATATTGGTGAACTTTG

cdsMIR156A_GG_L-1_R7 ttggtctcaacaaaagcATTTTAGAACTATCAATAGATTTGATGAG

cdsMIM156_GG_L-1_F1 ttggtctcaacataatgAAGAAAAATGGCCATCCCCTAGCTAG

cdsMIM156_GG_L-1_R1 ttggtctcaGTCCTCATTGCTCCATATCTTAAAACGC

cdsMIM156_GG_L-1_F2 ttggtctcaGGACTGCAGAAGGCTGATTCAGAC

cdsMIM156_GG_L-1_R2 ttggtctcaacaaaagcGAGGAATTCACTATAAAGAGAATCGG

Reverse transcription miR156 RT

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTCA

AtSnoR101 R1 AGCATCAGCAGACCAGTAGTT

Oligo dT TTTTTTTTTTTTTTTTTTTTT

qPCR ACT2-F GCACCCTGTTCTTCTTACCG

ACT2-R AACCCTCGTAGATTGGCACA

WUS F ACAAGCCATATCCCAGCTTCA

WUS R CCACCGTTGATGTGATCTTCA STM_qPCR_F ACAACTGCTTGATTGGTGGAGCC

STM_qPCR_R TCTGGTCCAGCCCCGTTGAT

CLV3_qPCR_F GTTCAAGGACTTTCCAACCGCAAGATGAT CLV3_qPCR_R CCTTCTCTGCTTCTCCATTTGCTCCAACC

FUL qPCR F3 TCCATATCTGCGCTCCAGAA

FUL qPCR R3 TGACCCGTTTTCTTCTCCCT pri-miR172bF CGGATTAGGGCGTTAATTACAATG

pri-miR172bR GGTCTCTGGACGAACTATTCTGTA

AP2 qPCR F AGTCAAGATATGCGGCTCAGGATGAAC AP2 qPCR R TCCGCTACCAATGTTGCTGCT

TOE1 qPCR F2 TGAGATTAACTCTGAGAGCAATAACT

TOE1 qPCR R2 CCCATGTATTCGTTATCTATCATT TOE2 qPCR F2 GTTCTTTTCACCCATGGAAAGAACAC

TOE2 qPCR R2 ACTGGACTGATCATGCCCTTGCCATG

SPL9 F GGAATTTGACCTAGAGAAAAGGAGTT SPL9 R GCATCACCATTTTCGTAAAGCGAAG

AtSnoR101 F1 CTTCACAGGTAAGTTCGCTTG

AtSnoR101 R1 AGCATCAGCAGACCAGTAGTT miR156 F GCGGCGGTGACAGAAGAGAGT

miRNA R GTGCAGGGTCCGAGGT

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