supplemental data supplemental protocol s1.€¦ · capture microdissected cell types from h....

SUPPLEMENTAL DATA

Supplemental Protocol S1. Laser capture microdissection, RNA isolation and amplification.

Butyl methyl methacrylate (BMM) was used as an embedding medium for structural

preservation of ovule tissues as specified in Materials and Methods. This medium has been used

routinely in cytological analyses, particularly for in situ hybridization in sexual and apomictic

Hieracium. The BMM matrix is readily removed with acetone prior to laser microdissection

(Baskin et al., 1992; Tucker et al., 2003; Okada et al., 2007; Tucker et al., 2012a).

Histological detail was well preserved after acetone treatment and individual cells such as AI

cells and other tissue layers remained relatively easy to identify prior to LCM (Figure S1A).

However, when the Leica laser was used under the conditions described in Materials and

Methods to dissect cell types it left a broad trace (Figure S1B-C) where adjoining cells were

considered to be destroyed as per other published experiments using LCM. This trace is

comparable to that previously reported in other studies dissecting larger tissue masses (Day et

al., 2005) but not as fine as the uniquely modified laser used by Wuest et al. (2010) to dissect

individual Arabidopsis mature female gametophyte cells. We conducted experiments to

examine if we were able to isolate RNA from distinct cell types in sufficient quantity for 454

sequencing analyses that would enable detection of low copy genes.

In preliminary LCM experiments, we examined the recovery of RNA when ovules were

excised from 5µm thick ovary sections by laser capture (Figure S1A; white dashed line). We

estimated that each captured ovule section contained 250 “cells”. When 20 ovule sections

containing approximately 5,000 “cells” were captured and the RNA extracted using a PicoPure

kit (Arcturus Bioscience Inc, Mountain View, CA, USA), the RNA recovery was low and

difficult to quantify (NanoDrop spectrophotometer, Thermo Scientific, Wilmington, DE,

USA). Therefore, the quantity of RNA was estimated by indirect measurement using RT-

PCR on known quantities of whole ovary RNA samples to detect the presence of a low level

ovary expressed gene MAP3K (ID 7.01 Table S2). The intensity of a band generated from

using RNA extracted from 20, and 2, captured ovule sections was compared to that generated

from a PCR reaction with 25ng, 5 ng and 1 ng of whole ovary input RNA (Figure S1D).

From this we estimated that the RNA isolated from 20 ovule sections was approximately 1–2

ng. As this equates to approximately 5,000 “cells”, we considered it was unrealistic to harvest

AI “cells” under these experimental conditions and obtain sufficient RNA for transcriptome

generation without amplification

In the next series of experiments, RNA extracted from 2 captured ovule sections ~500

“cells”) predicted to contain 0.1-0.2 ng RNA was amplified once using the MessageAmpTM

II

aRNA Kit (Ambion Inc, Austin, Texas, USA). Indirect measurement by RT-PCR using 2 μl

out of the total 100 μl of aRNA was equivalent to the 5 ng RNA input amplification control

indicating a total yield of 250 ng aRNA. We concluded that harvesting “100–250” individual AI

“cells” from 5 µm sections by laser capture would be sufficient material for RNA isolation,

amplification and use in downstream transcriptome experiments, particularly if a second

amplification step was included. Figure S1F shows that the amplified RNA was free of

genomic DNA contamination and Figure S1G shows that the Hieracium FIE gene was detected

in all samples as expected, while the HDMC1 gene expressed during meiosis in megaspore

mother cells was absent in the three samples. Remaining experiments focused on accumulating

captured cell samples and isolating RNA, amplification as described in Materials and Methods

and quantifying yields (Table S2).

Supplemental Figure S1. Validation of quality and quantity of amplified RNA from laser

capture microdissected cell types from H. praealtum ovule sections.

A, An ovary longitudinal section showing an ovule outlined in white and an AI cell

outlined in yellow before laser microdissection. B, The same section in A after

tracing around the AI cell with the laser. The pink shading indicates the damage path

of the laser. The intact AI cell is outlined in yellow. C, View of the section in B after

the AI cell was harvested into the cap of the capture tube. Bars = 50 µm. D,

Estimation of the quantity of RNA. RNA isolated from 20 (Lane1) and 2 (Lane 2)

captured whole ovule sections (5µm thick) was used for RT-PCR; Lane 3-5, Stage

4 ovary RNAs (25, 5 and 1 ng, respectively) were used as controls for quantity

estimation. E, Quantification of amplified RNA. RNA isolated from 2 whole ovule

sections was subjected to one round of RNA amplification followed by RT-PCR

using 2 µl of amplified product (Lane 1). Lane 2-4, Stage 4 ovary RNAs (5, 1 and

0.2 ng, respectively) were used as controls for quantity estimation. F, Assessment

of genomic DNA contamination in the amplified RNA samples. RT-PCR was

carried out using equal quantities of aRNA from sporophytic ovule (SO) cells,

aposporous initial (AI) and early aposporous embryo sac (EAEs) samples, and

genomic (g) DNA. G, Validation of aRNA quality derived from LCM-harvested cells

using Hieracium genes with known ovule cell-type expression profiles. Expression of

HDMC1 and HFIE in Hieracium ovules has been characterized by RT-PCR and in

situ hybridization (Okada et al., 2007; Rodrigues et al., 2008).

Supplemental Figure S2. Validation of RT-PCR data obtained for low ovary-expressed genes

in LCM samples by quantitative real-time PCR. The three AI cell detected genes (class II,

Figure 1C; ID 9.45 RD22, ID 24.04 NLR and ID 27.18 LOX, Table S2) and two genes from

class III (Figure 1C; ID 9.20 BAM1 and ID 9.10 CKX) and one from class IV (Figure 1C, ID

25.03 S-protein; Table S2) were chosen for quantitative PCR analysis. Gene expression levels

relative to the reference HUBQ gene (Rodrigues et al., 2008) are plotted on the y-axis for each

examined cell type. Abbreviations; sporophytic ovule (SO) cell, aposporous initial (AI) cell and

early aposporous embryo sacs (EAEs).

Supplemental Figure S3. Examination of two AI cell-detected genes, an abscisic acid-responsive

RD22-like gene and a CC-NBS-LRR-like disease resistance gene, by in situ hybridization in

ovules of sexual and apomictic plants. A,B,E and F, antisense RNA (AS) probes were hybridized

with apomictic (Apo) H. praealtum R35 ovary sections and sexual (Sex) H.pilosella P36 ovary

sections in D and H. In C and G, sense RNA (S) probe was hybridised with apomictic H.

praealtum R35 ovary sections as a negative control. The 9.45 RD22 RNA probe was applied in A-

D, and the 24.04 NLR RNA probe in E-H.

Abbreviations: AI, aposporous initial cell; DM, degenerating megaspores; EAE sac, early

aposporous embryo sac; FM, functional megaspore; NE, nucellar epidermal cells. Bars = 20 µm.

Supplemental Figure S4. Flow chart of bioinformatic analyses of RNA sequencing data from

each laser-captured ovule cell type (AI, aposporous initial; EAE sacs, early aposporous

embryo sac; SO cells, sporophytic ovule cells) with thresholds and filtering as shown. Three

in silico analysis approaches were used. A, sequence based analysis of assembled contigs. B,

Pfam annotation analysis of un-assembled sequence reads, median read length was 260 base

pairs. C, Analysis of gene ontology annotations of assembled contigs assigned through

sequence homology, median contig length was 453 base pairs.

Supplemental Figure S5. Comparison of the number of sequence reads in 15 genes

assembled de novo and their expression in the three LCM samples by quantitative real-time

PCR. For each gene (see Table S2 for gene IDs) the normalized sequence read number in

reads per kilobase per million (RPKM) is presented in the left panel (y-axis) and Q-PCR

expression measures relative to the control UBC9 gene are presented in the right panel (y-

axis). The SO cell 64 gene was not verified and the AI 1771 and SO 1226 genes only

partially verified following the suite of comparative control genes (see Methods).

Supplemental Figure S6. Radar plots showing the similarity between Arabidopsis and

apomictic H.praealtum ovule LCM data sets based on detection of TAIR10 transcripts on

Agilent 4x44K arrays. The Hieracium datasets consist of TAIR10 homologues of Hieracium

contigs. Gene lists were filtered to remove genes expressed in every dataset, and the values

represent the similarity of each list to the base dataset indicated in each panel. NUC,

Arabidopsis nucellus at meiosis, OV (early), Arabidopsis whole ovule at meiosis, OV (late),

Arabidopsis whole ovule at FG2-4 stage, FG2-4, Arabidopsis female gametophyte stage FG3-4,

SO cells, Hieracium sporophytic ovule cells, AI, Hieracium aposporous initial cells, EAE sac,

Hieracium early aposporous embryo sac at FG2.

Supplemental Figure S7. Differentially expressed Arabidopsis homologs of H.praealtum

contigs from AI cell, SO cell and EAE sac data sets. Red dots indicate relative expression in

the FG2-4 vs ovule (late) and blue dots indicate the relative expression in the nucellus vs whole

ovule (early).

Cell type a Ovule b SO cells AI cells c EAE sacs c

Number of “cells” 500 2,500 270 100 Original RNA d 0.1-0.2 ng 0.5-1 ng 0.1-0.2 ng 0.1-0.2 ng 1st amplification e 600 ng 1.3 µg 646 ng 513 ng 2nd amplification f

N.A 189 µg 154 µg 126 µg

f, 10% (10 µl) of the first round amplified material was used for the second amplification. N.A, not available.

Supplemental Table S1 . Isolation of RNA from laser captured H. praealtum R35 apomictic ovule cell types and RNA yields after amplification.

a, Whole ovules, clusters of somatic ovule (SO) cells, aposporous initial (AI) cells and 2-4 nucleate early aposporous embryo (EAE) sacs were collected by LCM from 5 µm sections. b, Two LCM ovule sections estimated to contain 500 "cells" in cross section were used for RNA isolation and amplification.

c, This represents the number of individual AI “cells” or EAE sacs cut from 5µm thick sections. d, Quantity of original RNA was estimated using an indirect PCR method to amplify a control gene from a known amount of ovary RNA. e, Quantity of RNA after the first round of amplification was determined using a Nanodrop spectrophotometer.

Identifier Protein E-value SO AI EAE Forward Reverse

2.07 AT1G74770.1 unknown protein 2.00E-47 GO673075 VIATTACATGACCAC

ACGCAAG

AGGTAACGAGGAA

AGGATTC

2.11 AT3G56740.1

ubiquitin-associated

(UBA)/TS-N domain-

containing protein

2.00E-37 GO673077 + + + IACAACCCCTCCG

ATTAATTG

GTAAGCACACTCA

AAGTTGG

2.13 AT4G37190.1 unknown protein 4.00E-33 GO673078 VIICCAAAACCCCAAT

TTCGAAG

TTTATCAGGTTTC

GTGTCCG

2.18 No hits GO673081 IIIAACAACCAAGACG

TTCCAAC

TTTGAAGCCTCTA

TGCTGTC

2.20 AT5G59380.1MBD6 (methyl-CpG-binding

domain 6)2.00E-04 GO673083 I

GGGCAATTCTGTC

ATTTGTC

CTGGACAGAAATT

CAGATCC

2.21 AT3G04380.1SUVR4; histone-lysine N-

methyltransferase3.00E-09 GO673084 I

TAGGAAAGCTCTA

ATGCTGC

GATGGAACATGCT

TATCCTG

4.06 AT3G62770.1

AtATG18a (Arabidopsis

thaliana homolog of yeast

autophagy 18 (ATG18) a)

2.00E-08 GO673086 IATAGATTGCGATC

AATGGGG

GATGACTCAACTG

GAATATC

4.07 AT2G27880.1 argonaute protein (AGO5) 3.00E-05 GO673087 VITTCCATTGACCAA

TGGATGG

AAGAGCGTAGAAG

GTGAAAG

4.09 AT4G21710.1

EMB1989, RPB2, NRPB2 |

NRPB2 (EMBRYO

DEFECTIVE 1989); DNA

binding

1.00E-88 GO673089 IGTACGATACCGAA

TACGTTC

AAGAACGTCTCTT

TGACCAG

4.12 AT1G68720.1cytidine/deoxycytidylate

deaminase family protein2.00E-25 GO673090 I

TGTAATGGGAAGA

CACGAAG

AGAAGCTAAAAAG

GGTGCTG

4.18 No hits GO673092 ITCGCGTATTTAAG

TCGTCTG

TTTTGATCCTTCG

ATGTCGG

4.19 No hits GO673093 ICATAGCTTTTAGA

AGACGCG

GACAGAAATTGGG

ACAGAAG

4.20 AT5G19300.1 expressed protein 2.00E-17 GO673094 IIITGGCTAAAAAGAA

GAGGGAC

GACCCACAAATCT

TAAGCTG

5.04 AT5G65700.1

BAM1 (big apical meristem

1); ATP binding / kinase/

protein serine/threonine

kinase

2.00E-52 GO673095 IIITACGAGTACATGA

GAAACGG

GAACATTAGCCAG

TCTAGAG

5.05 AT5G47870.1 unknown protein 5.00E-40 GO673096 ND Paste photo ND VTGCAAACCGCAAA

AAATCCC

ATCACGTATTTCT

CCACACC

Supplemental Table S2. Expression of low level Hieracium piloselloides (D18) ovary genes in amplified AI cell, SO cell and EAE

sac H. praealtum aRNA samples by RT-PCR.

Primers for RT-PCRe

Classd

BlastX Against Arabidopsis protein (TAIR database)a

ID No.Accession

No.b

LCM RT-PCRc

Table S2 (continued)

5.11 AT3G49500.1

SGS2, SDE1, RDR6 | RDR6

(RNA-DEPENDENT RNA

POLYMERASE 6); nucleic

acid binding

5.00E-23 GO673100 IAATCACGACTTCA

AAAGCGG

CGAGTTTGAGAAG

TTTTCGG

5.13 AT4G38170.1FRS9 (FAR1-related

sequence 9); zinc ion binding9.00E-52 GO673101 IV

TTACATAAACGCC

ACCACTC

ATGAGGAAGGAAG

TGTAAGG

5.14 AT3G60860.1guanine nucleotide exchange

family protein4.00E-20 GO673102 I

AAACGAACCCTGT

CTAACAC

CCAAATAACCCAA

AGCAGAC

5.16 AT3G49600.1

UBP26 (ubiquitin-specific

protease 26); ubiquitin-

specific protease

4.00E-44 GO673104 IAATGGGCATTCAC

CAAGATC

AAATCGGCGGTTA

TCCAAAC

7.01 AT3G13530.1

MAP3KE1, MAPKKK7 |

MAPKKK7 (MAP3K EPSILON

PROTEIN KINASE); kinase

1.00E-44 GO673105 + + + IAACGCGATTTCAA

GAAGCAG

AAGACGCGGTTCA

AAAACTC

7.02 AT2G28360.1SIT4 phosphatase-

associated family protein9.00E-57 GO673106 III

AATTTCTTCCGGA

ACCATCC

CTGTCTCTACTTC

TCTCTTG

9.01 AT5G49910.1

HSC70-7, cpHSC70-2 |

cpHSC70-2 (HEAT SHOCK

PROTEIN 70-7); ATP binding

/ unfolded protein binding

2.00E-40 GO673107 IIIATTCTAATCCAAG

AACGGCC

TGGGAATCGTTGA

AGTATGC

9.02 AT5G65700.1




kinase

5.00E-26 GO673108 ITCACTCTCCTCTT

CATATGC

AGCTGTAACAGGG

AGAAAAC

9.03 AT3G15010.1RNA recognition motif (RRM)-

containing protein5.00E-28 GO673109 III

AGAGAAAGACGGA

AGAGAAC

TTAACGCCAGAAT

TGTACCG

9.04 AT3G61250.1

AtMYB17 (myb domain

protein 17); DNA binding /

transcription factor

4.00E-31 GO673110 ITCCTAACACTGAG

TTCTAGG

CAAAATCCCAAAC

CTCCAAG

9.06 No hits GO673111 VCTCCATAATCAGT

CCATTCC

TCGGGTTTGTTTT

ACGAGAG

9.07 AT5G66680.1

DGL1 (defective glycosylation

1); dolichyl-

diphosphooligosaccharide-

protein glycotransferase

4.00E-59 GO673112 ITGGAGACAAGTTC

CATTTCC

ACAGGATCAATGA

TGACCTG

9.08 AT3G52260.1pseudouridine synthase

family protein 2.00E-38 GO673113 VI

CAACTCCATCTAA

TCCAACC

CAGCCATTGAGCA

AAAGTTC

9.09 AT4G11820.1

HMGS, MVA1, BAP1 | BAP1

(hydroxymethylglutaryl-CoA

synthase)

4.00E-50 GO673114 ITACAACATCACCC

CTTAACC

CAATTGGGGAACA

TGTACAC

9.10 AT1G75450.1

ATCKX5, ATCKX6, CKX5 |

CKX5 (CYTOKININ OXIDASE

5); cytokinin dehydrogenase

6.00E-16 GO673115 VTCAAATCGTGCCT

AACACTC

CATTGGATAATGG

GGAAGAG


9.11 AT5G42620.1metallopeptidase/ zinc ion

binding1.00E-19 GO673116 I

CATCAACACTTTG

TGCCATG

GCTGCTAAAAGAT

TAGCGTG

9.12 AT5G48930.1HCT | transferase family

protein9.00E-34 GO673117 IV

AAACCAACACCAA

ACCCATC

CACTTGAAACGTG

TAACCTG

9.14 AT5G50260.1 cysteine proteinase, putative 2.00E-55 GO673118 IIITGCATGGAGCTTT

GATTTCC

GTCGACTAGTTCT

TGTTCAG

9.15 AT2G39190.1ATATH8 | ATATH8 (ABC2

homolog 8)4.00E-31 GO673119 I

TAAACACTAATCC

TCACCGC

GATATTTCAAGAT

GGCGGAC

9.20 AT5G65700.1




kinase

2.00E-85 GO673121 IIICTCTGAAAAACCA

GTGTGTG

TGTTGTTGTACAG

GTCAAGG

9.21 AT1G60200.1

splicing factor PWI domain-

containing protein / RNA

recognition motif (RRM)-

containing protein

3.00E-13 GO673122 ITGTTAGGGAAAGA

GATGTGC

TCGTATCCGGATT

AATTGGG

9.22 AT5G03690.1fructose-bisphosphate

aldolase, putative5.00E-24 GO673123 III

TGATGAGCTTATT

GCCAACG

AGAGAGTTTCTTC

GAAGAGG

9.23 AT4G00740.1dehydration-responsive

protein-related2.00E-19 GO673124 III

AATGGGCGAGAGT

AAAAGAG

TCATTCATGGGGT

TGCTATG

9.25 AT1G60730.1aldo/keto reductase family

protein3.00E-29 GO673125 I

TCCTGAAACCATC

AGAAGAG

TCCGAGTTCGTAT

AACTGTG

9.27 AT3G11980.1 MS2 (MALE STERILITY 2) 5.0E-09 GO673127 IIICAACTTCACAACC

AACATCC

TTCTTTCCAACAT

CAGGCAC

9.29 AT2G44490.1

PEN2 (PENETRATION 2);

hydrolase, hydrolyzing O-

glycosyl compounds

7.00E-21 GO673128 VIIAAATGCTGGTGAT

CGTTTGC

AGCAACAATTCGT

AGGAGTC

9.30 AT1G61620.1protein binding / zinc ion

binding1.00E-30 GO673129 I

TACACACTGCGAA

TGTTTCG

AACGAGGAAAAGA

CGGATTC

9.34 AT5G66420.1 unknown protein 1.00E-48 GO673131 ITGAACTCATGATC

ACACGTG

TGGAGGTGGAGAT

GATAAAC

9.38 No hits GO673133 IGAAGTGCATTTCC

AGATCTG

ATGGCAACAGTTT

TGGCAAG

9.39 AT1G64550.1

ATGCN3 (Arabidopsis

thaliana general control non-

repressible 3)

7.00E-15 GO673134 IIIATTTCGCCCTATA

GTGAGTC

GGCCAATTTGGTA

AGGAAAG


9.42 AT2G23910.1cinnamoyl-CoA reductase-

related 8.00E-17 GO673135 V

CAACCAATGTTGT

TACACGG

GAACATGGTGTCC

ATAAACG

9.44 AT2G44950.1

HUB1 (HISTONE MONO-

UBIQUITINATION 1); protein

binding / zinc ion binding

5.00E-23 GO673136 IACCCGACAACAAA

CTTTCAC

CACTCAAAGGAGA

CTACAAG

9.45 AT5G25610.1RD22 (RESPONSIVE TO

DESSICATION 22)2.00E-34 GO673137 II

TTGTGGGATGTCA

TGCAATG

CAGCGTAGAGGTA

ATTGAAC

9.47 AT3G21280.1

UBP7 (UBIQUITIN-SPECIFIC

PROTEASE 7); ubiquitin-

specific protease

1.00E-52 GO673138 VTACATATCAATGG

TGCGAGC

AGTGGATATCCAT

TGGAGTG

24.02 AT4G23500.1

glycoside hydrolase family 28

protein / polygalacturonase

(pectinase) family protein

2.00E-55 GO673139 ICATCTTGGCTATC

GATTCTG

TCAACATCACTCG

AAATCCC

24.03 AT4G28300.1hydroxyproline-rich

glycoprotein family protein7.00E-34 GO673140 III

ACTAAAAATCTCT

CTCCCCG

TGATGTACGATAG

TGAACCG

24.04 AT3G14470.1disease resistance protein

(NBS-LRR class)2.00E-18 GO673141 II

ATTGTGTCTGCCT

TGTACTC

GCACGATGCTAAA

GTTTCTG

24.12 No hits GO673142 IGAAGAACGTAGCA

AAATGCG

CAAGACACACGAC

ACATGAC

25.03 AT4G16195.1self-incompatibility protein-

related 2.0E-05 GO673145 IV

CTCAAGTCTTGTC

CTGTATC

TTGCGCGGATTTG

GATTATC

25.04 AT1G08760.1

similar to unknown protein

[Arabidopsis thaliana]

(TAIR:AT4G13370.1)

8.0E-24 GO673146 IVATTGTGACCCACA

ACTATGC

AAAGGGGGCGAT

CTTTTATG

25.10 AT1G55320.2

similar to acyl-activating

enzyme 17 (AAE17)

[Arabidopsis thaliana]

(TAIR:AT5G23050.1); similar

to acyl-activating enzyme 18,

putative

2.0E-46 GO673147 ICGTAACTCCTCCT

TCAATTG

TATTTGGGAGCAA

CGGATAG

26.01 AT5G67360.1 ARA12; subtilase 2.0E-16 GO673148 VACACACACACTCA

CTAAAGC

TATTTCAACTCCG

GCAACAC

26.04 AT4G12710.1armadillo/beta-catenin repeat

family protein6.0E-18 GO673149 VII

CATTCTCGTCTTT

TGCACAG

TTATGGAAGGGAA

GATGCAC

27.01 AT5G55310.1TOP1alpha

(TOPOISOMERASE I)8.0E-35 GO673152 IV

AAGCCACCTGTCA

ATCATTC

TCATCTGACTCAG

ATTCTGG

27.06 No hits GO673154 ICACTCTGCCACTT

ACAATAC

GCAGAATTCACCA

AGTGTTG


27.09 No hits GO673155 IAAGTTAGTCTTCA

GCCACAC

AAACGGTAAGAAA

ACACGGC

27.13 AT5G52510.1scarecrow-like transcription

factor 8 (SCL8)7.0E-47 GO673156 IV

CCATCCACCCAAA

ACTAATC

TTATCCGATCTCA

ATACGGC

27.14 AT4G23160.1 protein kinase family protein 6.0E-28 GO673157 VITTCAATGCAAGCA

TTGTGGG

TTTGGGTGATCGA

CCTTTAG

27.17 AT4G16280.1 FCA (FCA); RNA binding 1.0E-04 GO673158 ICTAATGGAGATCA

ATCACGG

ACAACAGACACAG

AGTCTTG

27.18 AT3G45140.1 ATLOX2, LOX2 | LOX2

(LIPOXYGENASE 2)4.0E-56 GO673159 II

AACAACAGTACAC

ACTCGTG

AGGCACTCTTGAA

ATGCTTC

27.27 AT1G19100.1

ATP-binding region, ATPase-

like domain-containing

protein-related

8.0E-04 GO673160 IIITTTATAACACTCC

CCCTTGG

TTGGTCCAGAAGT

ACCATAC

27.29 AT3G07160.1

ATGSL10 (GLUCAN

SYNTHASE-LIKE 10); 1,3-

beta-glucan synthase

7.0E-37 GO673162 IACAACAAGTTGCA

AAGCCAG

ACGAACAAAATCG

GGAACAG

27.30 AT3G03940.1 protein kinase family protein 2.0E-19 GO673163 VGTACACTTTTCCC

TTACACC

GAAACACAGGAGA

CATTACG

27.34 AT3G23640.1

HGL1 (HETEROGLYCAN

GLUCOSIDASE 1);

hydrolase, hydrolyzing

O-glycosyl compounds

2.0E-48 GO673164 IIIGGTTTTTGAACCG

ATTCTGG

TTGTTGATAGCCC

AATGACC

27.35 AT1G68530.1

CER6, G2, POP1, CUT1 |

CUT1 (CUTICULAR 1);

acyltransferase

6.0E-38 GO673165 IACGAAGCAACAAC

GTGAAAG

GTTGAAGCATCCA

GAATGAC

27.37 AT1G03280.1

transcription initiation factor

IIE (TFIIE) alpha subunit

family protein / general

transcription factor TFIIE

family protein

2.0E-53 GO673166 IACATCTCTAGGAA

GAACACC

GTTCGGGAAATGT

GGATTTG

HUBQ AT4G05320.1 UBQ10NM_001084

884 I

ACTCCACTTGGTC

TTGCGTCT

AGTACGGCCGTC

TTCAAGC

HFIE (D36) AT3G20740.1

FIS3, FIE1, FIE | FIE

(FERTILIZATION-

INDEPENDENT

ENDOSPERM 1); nucleotide

binding / transcription factor

e-172 EU439051 ICCAGGAGAGGGC

ACAGTTGATA

GGGCTAGTTTGCA

ATTCCCATA


HRBR AT3G12280.1

RBR, RB, RBL1, RBR1 |

RBR1 (RETINOBLASTOMA-

RELATED 1)

0 EU439049 VICATGTGTTGGAGA

GAGCACACA

ACTTGATGAAGCG

GGACCTTTC

HDMC1 AT3G22880.1

DMC1, ATDMC1 | ATDMC1

(RECA-LIKE GENE); ATP

binding / DNA-dependent

ATPase/ damaged DNA

binding

e-163 EF530197 - - - -CAGCTGGCTCAC

ACTCTCTG

TCAAGTACAGCTC

CAGCATCC

a, The most similar Arabidopsis genes searched by blasts with TAIR database (http://www.arabidopsis.org/index.jsp) are listed with AGI identifier number, similarity and E-value.

b, Accession number of the Hieracium gene sequence deposited on GenBank database

c, RT-PCR anaylsis using aRNA from LCM samples. SO, Somatic ovule cells; AI, aposporous initial cells; EAE, embryo sac; -, no expression

d, Class defined by expression pattern (see text and Figure 1). -, no expression in any cell type

e, Primer sequences used for RT-PCR analysis

Pfam Domain ID Pfam Domain GO ID GO Description Ontologya Number in

input listb

Number in

BG/Refb p-value

c

SO vs AI

Enriched in SO

PF03095 PTPA Phosphotyrosyl phosphate activator (PTPA) protein GO:0019211 phosphatase activator activity F 5 0 3.2E-03

PF03953 Tubulin_C Tubulin C-terminal domain GO:0003924 GTPase activity F 22 8 1.0E-03

PF00230 MIP Major intrinsic proteins GO:0005215 transporter activity F 23 9 1.3E-03

PF02984 Cyclin_C Cyclin GO:0005634 nucleus C 11 3 3.4E-03

PF01370 Epimerase NAD dependent epimerase/dehydratase family GO:0003824 catalytic activity F 8 2 3.3E-03

PF00657 Lipase_GDSL GDSL-like Lipase/Acylhydrolase GO:0016788 hydrolase activity, acting on ester bonds F 6 1 3.8E-03

PF01915 Glyco_hydro_3_C Glycoside hydrolase family 3 GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds F 5 1 4.2E-03

PF08241 Methyltransf_11 Methyltransferase domain GO:0008168 methyltransferase activity F 5 1 4.2E-03

Enriched in AI

PF03931 Skp1_POZ Skp1 family, tetramerisation domain GO:0006511 ubiquitin-dependent protein catabolic process P 11 3 7.4E-04

PF00240 ubiquitin Ubiquitin GO:0005515 protein binding F 57 38 1.4E-03

PF08148 DSHCT DSHCT (NUC185) domain GO:0005524 ATP binding F 7 1 2.0E-03

PF00298 Ribosomal_L11 Ribosomal protein L11, RNA binding domain GO:0003735 structural constituent of ribosome F 12 4 2.7E-03

PF01466 Skp1 Skp1 family, dimerisation domain GO:0006511 ubiquitin-dependent protein catabolic process P 15 6 2.8E-03

PF01655 Ribosomal_L32e Ribosomal protein L32 GO:0003735 structural constituent of ribosome F 16 7 4.3E-03

AI vs EAE

Enriched in EAE

PF01095 Pectinesterase Pectinesterase (EC 3.1.1.11) GO:0030599 pectinesterase activity F 6 2 2.3E-03

PF00112 Peptidase_C1 Cysteine protease GO:0008234 cysteine-type peptidase activity F 11 7 2.3E-03

PF00235 Profilin Profilin GO:0003779 actin binding F 5 2 4.1E-03

Enriched in AI

PF00400 WD40 WD40 repeat GO:0005515 protein binding F 84 27 2.7E-03

Supplemental Table S3. Pfam enrichment analysis by reciprocal and pairwise comparisons between H. praealtum ovule cell types.


SO vs EAE

Enriched in SO

PF00400 WD40 WD40 repeat GO:0005515 protein binding F 82 27 3.3E-03

PF00230 MIP Major intrinsic proteins GO:0005215 transporter activity F 23 5 7.3E-03

PF03953 Tubulin_C Tubulin C-terminal domain GO:0003924 GTPase activity F 22 5 1.0E-02

PF01501 Glyco_transf_8 Glycosyl transferase family 8 GO:0016757 transferase activity, transferringGlycosylGroups F 11 1 6.8E-03

PF02535 Zip ZIP Zinc transporter GO:0046873 metal ion transmembrane transporter activity F 6 0 7.3E-03

PF03095 PTPA Phosphotyrosyl phosphate activator (PTPA) protein GO:0019211 phosphatase activator activity F 5 0 7.3E-03

PF01915 Glyco_hydro_3_C Glycoside hydrolase family 3 GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds F 5 0 7.3E-03

PF08241 Methyltransf_11 Methyltransferase domain GO:0008168 methyltransferase activity F 5 0 7.3E-03

Enriched in EAE

PF01599 Ribosomal_S27 Ribosomal protein S27a GO:0003735 structural constituent of ribosome F 12 11 3.3E-03

PF03719 Ribosomal_S5_C Ribosomal protein S5, C-terminal domain GO:0003735 structural constituent of ribosome F 11 10 5.7E-03

PF01780 Ribosomal_L37ae Ribosomal L37ae protein GO:0003735 structural constituent of ribosome F 11 9 1.3E-03

PF01095 Pectinesterase Pectinesterase (EC 3.1.1.11) GO:0030599 pectinesterase activity F 6 4 4.5E-03

PF00366 Ribosomal_S17 Ribosomal protein S17 GO:0003735 structural constituent of ribosome F 6 4 4.5E-03

PF00164 Ribosomal_S12 Ribosomal protein S12 GO:0003735 structural constituent of ribosome F 9 7 6.1E-03

PF05162 Ribosomal_L41 Ribosomal L41 GO:0003735 structural constituent of ribosome F 9 7 6.1E-03


PF00338 Ribosomal_S10 Ribosomal protein S10p/S20e GO:0003735 structural constituent of ribosome F 7 4 6.8E-04

PF02298 Cu_bind_like Plastocyanin-like domain GO:0005507 copper ion binding F 8 5 2.5E-03

PF00112 Peptidase_C1 Cysteine protease GO:0008234 cysteine-type peptidase activity F 11 7 2.5E-03

PF00067 p450 Cytochrome P450 GO:0005506 iron ion binding F 8 4 3.0E-03

PF04573 SPC22 Signal peptidase subunit GO:0008233 peptidase activity F 6 1 8.5E-04

PF00120 Gln-synt_C Glutamine synthetase (EC 6.3.1.2) GO:0004356 glutamate-ammonia ligase activity F 6 1 8.5E-04


a, GO functional categories. P, Biological process; F, Molecular function; C, Cellular Component

b, Total numbers of Pfam domains used in input or background reference for each cell type are 38,667 (SO), 42,962 (AI) and 17,924 (EAE).

c, Enrichment of Pfam domains was analysed by Fisher test (p<0.05).

GO term Ontologya Description

Number in

input listb

Number in

BG/Refb p-value

c

SO vs AI

Enriched in SO

GO:0006468 P protein amino acid phosphorylation 133 72 5.6E-03

GO:0007166 P cell surface receptor linked

signaling pathway

61 27 3.9E-02

GO:0007167 P enzyme linked receptor protein

signaling pathway

57 25 3.9E-02

GO:0007169 P transmembrane receptor protein

tyrosine kinase signaling pathway

57 25 3.9E-02

GO:0023052 P signaling 486 390 3.9E-02

GO:0023034 P intracellular signaling pathway 78 41 5.0E-02

GO:0004672 F protein kinase activity 168 94 4.0E-04

GO:0016773 F phosphotransferase activity, alcohol

group as acceptor

203 130 2.6E-03

GO:0016798 F hydrolase activity, acting on glycosyl

bonds

78 39 1.5E-02

GO:0004674 F protein serine/threonine kinase

activity

122 73 1.5E-02

GO:0016772 F transferase activity, transferring

phosphorus-containing groups

444 359 2.4E-02

GO:0004553 F hydrolase activity, hydrolyzing O-

glycosyl compounds

71 36 2.4E-02

GO:0004871 F signal transducer activity 184 130 2.9E-02

GO:0060089 F molecular transducer activity 184 130 2.9E-02

GO:0016301 F kinase activity 380 303 2.9E-02

GO:0005975d

P carbohydrate metabolic process 14 36.9E-03

GO:0008168d

F methyltransferase activity 5 0 3.3E-02

Enriched in AI

GO:0006412 P translation 995 746 1.7E-06

GO:0042254 P ribosome biogenesis 259 177 2.6E-02

GO:0022613 P ribonucleoprotein complex

biogenesis

259 177 2.6E-02

GO:0010467 P gene expression 1466 1260 2.6E-02

GO:0044267 P cellular protein metabolic process 1714 1501 3.2E-02

GO:0003735 F structural constituent of ribosome 724 533 3.3E-05

GO:0005198 F structural molecule activity 860 671 3.2E-04

GO:0009908d

P flower development 11 3 2.5E-02

GO:0048229d

P gametophyte development 5 0 2.9E-02

GO:0051704d

P multi-organism process 19 9 3.6E-02

GO:0010876d

P lipid localization 8 2 5.0E-02

AI vs EAE

There are no significant GO terms found to be enriched in this comparison

Supplemental Table S4. GO enrichment analysis by reciprocal and pairwise comparisons between

H. praealtum ovule cell types.


SO vs EAE

Enriched in SO

GO:0050789 P regulation of biological process 776 329 7.3E-03

GO:0019219 P regulation of nucleobase,

nucleoside, nucleotide and nucleic

acid metabolic process

204 64 7.3E-03

GO:0019222 P regulation of metabolic process 290 101 7.3E-03

GO:0006350 P transcription 420 162 7.3E-03

GO:0051171 P regulation of nitrogen compound

metabolic process

210 66 7.3E-03

GO:0060255 P regulation of macromolecule

metabolic process

265 91 7.3E-03

GO:0045449 P regulation of transcription 195 62 7.8E-03

GO:0065007 P biological regulation 908 401 7.8E-03

GO:0006468 P protein amino acid phosphorylation 133 37 8.3E-03

GO:0010468 P regulation of gene expression 256 89 8.3E-03

GO:0080090 P regulation of primary metabolic

process

227 77 9.0E-03

GO:0031323 P regulation of cellular metabolic

process

239 83 1.1E-02

GO:0050794 P regulation of cellular process 693 299 1.1E-02

GO:0023033 P signaling pathway 96 24 1.2E-02

GO:0009889 P regulation of biosynthetic process 218 75 1.2E-02

GO:0031326 P regulation of cellular biosynthetic

process

218 75 1.2E-02

GO:0010556 P regulation of macromolecule

biosynthetic process

209 72 1.5E-02

GO:0023052 P signaling 486 201 1.5E-02

GO:0023034 P intracellular signaling pathway 78 19 2.9E-02

GO:0043412 P macromolecule modification 579 253 4.2E-02

GO:0007167 P enzyme linked receptor protein

signaling pathway

57 12 4.2E-02

GO:0007169 P transmembrane receptor protein

tyrosine kinase signaling pathway

57 12 4.2E-02

GO:0032774 P RNA biosynthetic process 125 39 4.2E-02

GO:0016740 F transferase activity 949 397 2.0E-04

GO:0016773 F phosphotransferase activity, alcohol

group as acceptor

203 59 7.6E-04

GO:0016772 F transferase activity, transferring

phosphorus-containing groups

444 166 7.9E-04

GO:0004672 F protein kinase activity 168 48 1.8E-03

GO:0016301 F kinase activity 380 142 2.3E-03

GO:0003700 F transcription factor activity 215 70 3.3E-03

GO:0030528 F transcription regulator activity 299 111 9.6E-03

GO:0004674 F protein serine/threonine kinase

activity

122 36 2.2E-02

GO:0004871 F signal transducer activity 184 65 4.3E-02

GO:0060089 F molecular transducer activity 184 65 4.3E-02

GO:0009057d

P macromolecule catabolic process 23 4 2.6E-02

GO:0003824d

F catalytic activity 144 53 1.7E-02

GO:0016787d

F hydrolase activity 42 11 2.9E-02


Enriched in EAE

GO:0006412 P translation 630 746 2.9E-15

GO:0019538 P protein metabolic process 1092 1698 7.4E-05

GO:0044267 P cellular protein metabolic process 981 1501 7.4E-05

GO:0010467 P gene expression 840 1260 7.4E-05

GO:0042254 P ribosome biogenesis 161 177 2.0E-04

GO:0022613 P ribonucleoprotein complex

biogenesis

161 177 2.0E-04

GO:0034645 P cellular macromolecule biosynthetic

process

847 1316 1.7E-03

GO:0009059 P macromolecule biosynthetic

process

850 1324 1.8E-03

GO:0044249 P cellular biosynthetic process 1005 1619 1.0E-02

GO:0009058 P biosynthetic process 1117 1824 1.4E-02

GO:0008152 P metabolic process 1944 3342 4.0E-02

GO:0006091 P generation of precursor metabolites

and energy

159 207 4.2E-02

GO:0003735 F structural constituent of ribosome 477 533 2.0E-14

GO:0005198 F structural molecule activity 570 671 2.0E-14

GO:0010876d

P lipid localization 6 2 1.9E-02

GO:0051704d

P multi-organism process 11 9 3.5E-02

GO:0048229d

P gametophyte development 3 0 3.7E-02

GO:0008289d

F lipid binding 5 2 4.4E-02

GO:0005840d

C ribosome 65 89 6.4E-03

GO:0022626d

C cytosolic ribosome 63 86 7.0E-03

GO:0030529d

C ribonucleoprotein complex 68 95 7.3E-03

GO:0015934d

C large ribosomal subunit 37 44 9.8E-03

GO:0033279d

C ribosomal subunit 59 81 1.0E-02

GO:0043232d

C intracellular non-membrane-bounded organelle79 117 1.1E-02

GO:0022625d

C cytosolic large ribosomal subunit 36 44 1.4E-02

GO:0032991d

C macromolecular complex 94 152 3.0E-02

d, Identification by post-hoc GO enrichment analysis using nested GO (nEASE) analysis

a, GO functional categories. P, Biological process; F, Molecular function; C, Cellular Component

b, Numbers of contigs with GO term annotation used in input or background reference for each cell

type are 4032 (SO), 3896 (AI) 1820 (EAE), see Table III.

c, Enrichment of GO terms was analysed by Fisher test (p<0.05) with FDR adjustment using AgriGO.

Gene name TAIR10 ID

SPOROCYTELESS AT4G27330

DISRUPTION OF MEIOTIC CONTROL 1 AT3G22880

SPORULATION 11 AT1G63990

RAD50 AT2G31970

ASYNAPTIC 1 AT1G67370

MUTS HOMOLOG 4 AT4G17380

SOLO DANCERS AT1G14750

SWITCH1/DYAD AT5G51330

MULTIPOLAR SPINDLE 1 AT5G57880

MUTL-HOMOLOGUE 1 AT3G24320

REC8 AT5G05490

PARTING DANCERS AT1G12790

TARDY ASYNCHRONOUS MEIOSIS AT1G77390

CHROMATIN-REMODELING PROTEIN 11 AT3G06400

pFM AT4G12250

MNEME AT5G39840

MtN3 AT5G40260

MtN3 AT5G23660

ARABINOGALACTAN PROTEIN 18 AT3G11700

Supplemental Table S5 Arabidopsis meiosis and megaspore

associated genes examined for presence in H. praealtum AI cell contigs.

Gene Name

Comparison GO term (P<0.05) TAIR10 ID AI EAE SO Nuc Ov ES Ov

Enriched in AI and EAE v SO

flower development AT1G52740* ● ● ● ● ● HISTONE H2A PROTEIN 9 (HTA9)

AT1G79000* ● ● ● ● ● HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY 1 (HAC1)

AT2G40080 ● ● ● ● ● EARLY FLOWERING 4 (ELF4)

AT4G24960* ● ● ● ● HVA22 HOMOLOGUE D (HVA22D)

AT5G56030 ● ● ● ● ● HEAT SHOCK PROTEIN 81-2 (HSP81-2)

AT4G09960 ● ● ● ● ● SEEDSTICK (STK)

AT5G17690 ● ● ● ● ● TERMINAL FLOWER 2 (TFL2)

AT5G16780 ● ● ● ● DEFECTIVELY ORGANIZED TRIBUTARIES 2 (DOT2)

AT4G18960 ● ● ● ● ● ● AGAMOUS (AG)

AT2G38810 ● ● ● ● ● HISTONE H2A 8 (HTA8)

gametophyte development AT1G60490 ● ● ● ● VACUOLAR PROTEIN SORTING 34 (VPS34)

AT2G20490 ● ● ● ● ● ● NOP10, EDA27

AT4G24960* ● ● ● ● HVA22 HOMOLOGUE D (HVA22D)

AT2G35940* ● ● BEL1-LIKE HOMEODOMAIN 1 (BLH1)

AT2G47470* ● ● ● ● ● UNFERTILIZED EMBRYO SAC 5 (UNE5)

AT3G48750 ● ● ● ● CELL DIVISION CONTROL 2 (CDC2)

multi-organism process AT1G52740* ● ● ● ● ● HISTONE H2A PROTEIN 9 (HTA9)

AT3G03300 ● ● ● ● ● DICER-LIKE 2 (DCL2)

AT1G09770 ● ● ● ● ● ● CELL DIVISION CYCLE 5 (CDC5)

AT3G44750 ● ● ● ● ● HISTONE DEACETYLASE 3 (HDA3)

AT1G79000* ● ● ● ● ● HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY 1 (HAC1)

AT4G00860 ● ● ● ● ● ● ATOZI1

AT3G51300 ● ● ● ● ● RHO-RELATED PROTEIN FROM PLANTS 1 (ROP1)

AT5G50320 ● ● ● ● ● ELONGATA 3 (ELO3)

AT3G59760 ● ● ● ● ● O-ACETYLSERINE (THIOL) LYASE ISOFORM C (OASC)

AT5G01650 ● ● ● ● ● TAUTOMERASE MIF SUPERFAMILY PROTEIN

AT2G47470* ● ● ● ● ● UNFERTILIZED EMBRYO SAC 5 (UNE5)

AT2G35940* ● ● BEL1-LIKE HOMEODOMAIN 1 (BLH1)

AT5G55390 ● ● ● ● ● ENHANCED DOWNY MILDEW 2 (EDM2)

AT3G16640 ● ● ● ● ● ● TRANSLATIONALLY CONTROLLED TUMOR PROTEIN (TCTP)

AT5G33340 ● ● CONSTITUTIVE DISEASE RESISTANCE 1 (CDR1)

AT1G48410 ● ● ● ● ● ARGONAUTE 1 (AGO1)

AT3G17220 PECTIN METHYLESTERASE INHIBITOR 2 (PMEI2)

lipid localisation AT1G03103 ●

AT5G38170 ● ●

AT2G18370 ● ●

AT3G18280 ● ● ●

AT3G51590 ● LIPID TRANSFER PROTEIN 12 (LTP12)

AT5G64080 ● ● ● ● XYLOGEN PROTEIN 1 (XYP1)

AT1G43666 ●

AT1G48750 ● ● ●

Enriched in SO v AI

carbohydrate metabolic process AT1G66980 ● ● ● ● SUPPRESSOR OF NPR1-1 CONSTITUTIVE 4 (SNC4)

AT1G79550 ● ● ● ● ● PHOSPHOGLYCERATE KINASE (PGK)

AT3G52990 ●

AT2G01290 ● ● ● ● ● RIBOSE-5-PHOSPHATE ISOMERASE 2 (RPI2)

AT4G38270 ● ● ● ● ● GALACTURONOSYLTRANSFERASE 3 (GAUT3)

AT1G80160 ● GLYOXYLASE I 7 (GLYI7)

AT5G55700 ● ● ● ● ● BETA-AMYLASE 4 (BAM4)

AT5G64380 ● ● ● ●

AT5G24400 ● ● ● ● ● EMBRYO DEFECTIVE 2024 (emb2024)

AT5G64570 ● ● ● ● ● BETA-D-XYLOSIDASE 4 (XYL4)

AT3G61490 ● ● ● ● ●

AT4G36890 ● ● ● ● ● IRREGULAR XYLEM 14 (IRX14)

methyltransferase activity AT5G26880 ● ● ● ● ● AGAMOUS-LIKE 26 (AGL26)

AT5G15380 ● ● ● DOMAINS REARRANGED METHYLASE 1 (DRM1)

AT3G12270 ● ● ● ● ● PROTEIN ARGININE METHYLTRANSFERASE 3 (PRMT3)

AT5G13710 ● ● ● ● ● STEROL METHYLTRANSFERASE 1 (SMT1)

AT4G25730 ● ● ● ● ●

Supplemental Table S6. Discriminatory gene annotations associated with significant GO terms found through nested GO enrichment analysis between H. praealtum ovule cell types and comparison

with Arabidopsis ovule genes on Agilent 4x44k arrays. Blue dot (●) indicates detectable expression, gray identifies genes not present on the array, asterisk (*) highlights genes contributing to more than 1 GO

term enrichment in this list.

HieraciumArabidopsis

(Meiosis)

Arabidopsis

(FG 2-4)

supplemental data supplemental protocol s1.€¦ · capture microdissected cell types from h....

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