Charophyta - Plant Physiology · Mimulus guttatus A 72 89 Capsicum annuum 72 89 Mimulus guttatus B...

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At Zm Pp Mp 301 279 283 330 Land Plants Charophyta A B C Arabidopsis thaliana 100 100 Arabidopsis lyrata 97 99 Capsella rubella 96 98 Chorispora bungeana 95 97 Thlaspi arvense 95 98 Thellaungiella halopila 94 97 Brassica rapa 91 98 Carica papaya 80 91 Prunus persica 80 91 Populus trichocarpa B 79 84 Hevea brasiliensis 78 92 Malus domestica C 78 91 Manihot esculenta 78 92 Malus domestica A 78 92 Camptotheca acuminata 78 91 Lactuca serriola 77 90 Fragaria vesca 77 91 Ricinus communis 76 91 Citrus clementina 76 90 Citrus sinensis 76 90 Malus domestica B 76 ** Cannabis sativa 76 90 Cucumis sativus 76 88 Vitis vinifera 75 91 Eucalyptus grandis 75 90 Populus trichocarpa A 74 91 Theobroma cacao 74 91 Sesamum indicum 72 92 Gossypium raimondii 72 91 Mimulus guttatus A 72 89 Capsicum annuum 72 89 Mimulus guttatus B 72 89 Nicotiana benthamiana 72 89 Solanum lycopersicum 72 89 Cicer arietinum 70 87 Glycin max A 70 86 Glycin max B 70 87 Linum usitatissimum 69 87 Phaseolus vulgaris 68 86 Medicago truncatula 67 84 Utricularia gibba 66 86 Aquilegia coerulea 65 88 Genlisea aurea 64 88 Oryza sativa 60 87 Panicum virgatum 59 87 Costus pictus 59 88 Setaria italica 59 87 Zea mays 59 86 Musa acuminata 59 86 Brachypodium distachyon 59 87 Sorghum bicolor 59 87 Hordeum vulgare 58 86 Curcuma longa 57 87 Aegilops tauschii 57 86 Marchantia polymorpha B 50 80 Sellaginella moellendorffii A 48 82 Sellaginella moellendorffii B 48 82 Marchantia polymorpha A 47 78 Ceratodon purpureus 38 83 Physcomitrella patens 38 83 Bryophytes and lycophytes Monocots Dicots Supplemental Figure S1. Bioinformatic analyses of Loop sequences. A, Analysis of MEM domain topography of representative streptophyte DEK1 proteins predicted using the HMMTOP2.0 server. Each box (blue) represents a transmembrane segment (TMS). The green lines show the localization of the predicted Loop. B, Heatmap showing the degree of amino acid sequence identity between the various Loop and CysPc sequences. Numbering is % identity relative to Arabidopsis thaliana Loop sequence. **Partial CysPc sequence eliminated from the analysis. C, Graphical representation (WebLogo3) of land plant Loop sequence alignment (MAFFT) showing the conserved regions in the sequences. The amino acids are colored according to their chemical properties: polar amino acids = green, basic = blue, acidic = red and hydrophobic = black.

Transcript of Charophyta - Plant Physiology · Mimulus guttatus A 72 89 Capsicum annuum 72 89 Mimulus guttatus B...

Page 1: Charophyta - Plant Physiology · Mimulus guttatus A 72 89 Capsicum annuum 72 89 Mimulus guttatus B 72 89 Nicotiana benthamiana 72 89 Solanum lycopersicum 72 89 ... Genlisea aurea

At

Zm

Pp

Mp

301

279

283

330

Nitella mirabilis

Mougeotia scalaris

Klebsormidium flaccidum

295

229

370

Land Plants

Charophyta

A B

C

Arabidopsis thaliana 100 100Arabidopsis lyrata 97 99Capsella rubella 96 98Chorispora bungeana 95 97Thlaspi arvense 95 98Thellaungiella halopila 94 97Brassica rapa 91 98Carica papaya 80 91Prunus persica 80 91Populus trichocarpa B 79 84Hevea brasiliensis 78 92Malus domestica C 78 91Manihot esculenta 78 92Malus domestica A 78 92Camptotheca acuminata 78 91Lactuca serriola 77 90Fragaria vesca 77 91Ricinus communis 76 91Citrus clementina 76 90Citrus sinensis 76 90Malus domestica B 76 **Cannabis sativa 76 90Cucumis sativus 76 88Vitis vinifera 75 91Eucalyptus grandis 75 90Populus trichocarpa A 74 91Theobroma cacao 74 91Sesamum indicum 72 92Gossypium raimondii 72 91Mimulus guttatus A 72 89Capsicum annuum 72 89Mimulus guttatus B 72 89Nicotiana benthamiana 72 89Solanum lycopersicum 72 89Cicer arietinum 70 87Glycin max A 70 86Glycin max B 70 87Linum usitatissimum 69 87Phaseolus vulgaris 68 86Medicago truncatula 67 84Utricularia gibba 66 86Aquilegia coerulea 65 88Genlisea aurea 64 88Oryza sativa 60 87Panicum virgatum 59 87Costus pictus 59 88Setaria italica 59 87Zea mays 59 86Musa acuminata 59 86Brachypodium distachyon 59 87Sorghum bicolor 59 87Hordeum vulgare 58 86Curcuma longa 57 87Aegilops tauschii 57 86Marchantia polymorpha B 50 80Sellaginella moellendorffii A 48 82Sellaginella moellendorffii B 48 82Marchantia polymorpha A 47 78Ceratodon purpureus 38 83Physcomitrella patens 38 83B

ryop

hyte

s an

d ly

coph

ytes

M

onoc

ots

Dic

ots

Supplemental Figure S1. Bioinformatic analyses of Loop sequences. A, Analysis of MEM domain

topography of representative streptophyte DEK1 proteins predicted using the HMMTOP2.0 server. Each

box (blue) represents a transmembrane segment (TMS). The green lines show the localization of the

predicted Loop. B, Heatmap showing the degree of amino acid sequence identity between the various Loop

and CysPc sequences. Numbering is % identity relative to Arabidopsis thaliana Loop sequence. **Partial

CysPc sequence eliminated from the analysis. C, Graphical representation (WebLogo3) of land plant Loop

sequence alignment (MAFFT) showing the conserved regions in the sequences. The amino acids are colored

according to their chemical properties: polar amino acids = green, basic = blue, acidic = red and

hydrophobic = black.

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Supplemental Figure S2. Conserved domains detected in the Physcomitrella patens DEK1

protein by RPS-BLAST using the Conserved Domain Architecture Retrieval Tool (CDART)

at NCBI. PMT_2 Superfamily: cl19158, Dolichyl-phosphate-mannose-protein

mannosyltransferase; MFS: cl18950, The Major Facilitator Superfamily (MFS) is a large

and diverse group of secondary transporters that includes uniporters, symporters, and

antiporters; The calpain domains CysPc and Calpain_III (C2L).

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3´ TGS

pBHRF-DEK1-ΔLoop5´ TGS

Locus PpDEK1

loxP-35S-hptII-loxP5

5

6

6 7

8

8

9

9

10

10

5 6 8 9 10Locus PpDEK1-ΔLoop

A

5 6 8 9 10Locus PpDEK1-ΔLoop

pCR-PpLoop_V1 5 6 7 8 9 10

RT-Loop-F RT-Loop-R

Cre-mediated excision

B

5 6 8 9 10Locus PpDEK1-ΔLoop

108 95 6 7 MpDEK1-Loop 7 loxP-35S-nptII-loxP

5 6 7 ZmDEK1-Loop 7

5 6 7 AtDEK1-Loop 7

108 95 6 7 Mp/Zm/AtDEK1-Loop 7

pBN

RF-

DEK

1-Δ

Loop

Com

p co

nstru

cts

Locus PpDEK1-Loop Comp

RT-Loop-F RT-Loop-R

Cre-mediated excision

loxP-35S-hptII-loxP5 6 8 9 10Locus PpDEK1-ΔLoop before Cre

Locus PpDEK1-PpLoop Comp 5 6 7 8 9 10

5 6 7 Mp/Zm/AtDEK1-Loop 7

Locus PpDEK1-Loop Comp before Cre

35S-R Ter-F

Loop-Genot-F Loop-Genot-R

Loop-Genot-F Loop-Genot-RRT-Loop-F RT-Loop-R

Loop-Genot-F Loop-Genot-R

108 9loxP-35S-nptII-loxP

108 9loxP-35S-nptII-loxP

108 9loxP-35S-nptII-loxP

35S-R Ter-F

C

Supplemental Figure S3. Vector construction for targeted deletion and replacements of the PpLoop. A, Schematic

representation of the Loop deletion and elimination of the resistance cassette by Cre-mediated excision. The 5`and

3`targeting sequences (TGS) are boxed. The red box represent a 47 bp long sequence of the intron6-exon7 border

which was added to the 5`end of 3` TGS to avoid splicing conflicts with heterologous Loop sequences. B,

Schematic representation of the Loop replacements, knock-in of the native PpLoop sequence to the dek1Δloop

locus. C, Knock-in of the M. polymorpha (Mp), Z. mays (Zm) and A. thaliana (At) Loop-coding sequences to the

dek1Δloop locus and subsequent elimination of the resistance cassette by Cre-mediated excision. The numbering

in the boxes correspond to the exon number of the P. patens dek1 gene. Annealing sites for primers used for

genotyping are shown with arrow heads and the primer sequences can be found in Supplemental Table S2.

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Supplemental Figure S4. Gametophore morphology in dek1Δloop line before and after the Cre recombinase-

-mediated removal of the resistance cassette. A, Young WT gametophore. B, Developmentally arrested buds

of the dek1Δloop line before CRE recombinase treatment (arrows). C, Aberrant gametophore of the dek1Δloop

line after the Cre recombinase removal of the resistance cassette. Bar: 500 μm.

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dek1Δ

loop

Δdek1

WTWTM23130

9416

6557

4361

23222027

A

PpD

EK1-

Loop

Pro

be

B C

D

M1 M2 WT de

k1Δlo

op

MpLoo

p

AtLoop

ZmLoop

NC

DEK

1Tu

bulin

1000500

1001000500

100

5 6 7 8 9 10432 11

BglII BglII BglIIPpDEK1-Loop Probe (1.3 kb)

3.8 kb

WTΔd

ek1AtLoo

p#14

AtLoop

MpLoo

p#29

ZmLoop#

5

PpLoo

p

G41

8 Pr

obe

9.4

6.6

4.4

2.32.0

0.5

WT Δd

ek1AtLoo

p#14

AtLoop

MpLoo

p#29

ZmLoop#

5

PpLoo

p

5TG

S+3T

GS

Prob

e

9.4

6.6

4.4

2.32.0

0.5

(i)

5 TGS Probe (1 kb) 3 TGS Probe (1.2 kb)G418 Probe (0.8 kb)7 DEK1-Loop 7 loxP-35S-nptII-loxP5 6432

BglII

8 9 10 11

BglII BglIIBglII

(BglII)2.7 kb (MpDEK1-Loop only)

4.9 kb

0.8 kb2.1 kb (MpDEK1-Loop only) 0.6 kb

(BglII)

(ii)

Supplemental Figure S5. Southern blot genotyping, RT-PCR. A, Southern blot genotyping performed to confirm the absence of the Loop sequence in the dek1Δloop line after Cre/LoxP removal of theresistance cassette. Restriction fragments were generated using BglII and the blot was hybridized with the PpDEK1-Loop Probe displayed to the expected hybridizing fragment shown in (i). Positive control: WT; negative control: Δdek1. B, Southern blot genotyping of the PpLoop and the heterologous Loop replacement lines (At/Zm/MpLoop) using a mixture of the 5`TGS and 3`TGS probes. BglII was used to create the restriction fragments and expected hybridizing fragments are shown in (ii). C, Southern blot genotyping of the PpLoop and the heterologous Loop replacement lines (At/Zm/MpLoop) using the G418 probe. BglII was used to create the restriction fragments and expected hybridizing fragments are shown in (ii). The MpLoop line contains two additional BglII sites (shown in brackets, (ii)) that are absent in the AtLoop and ZmLoop replacement lines. The numbering in the boxes in (i) and (ii) corresponds to the exon number of the P. patens DEK1 gene. D, Semi-quantitative RT-PCR analysis of PpDEK1 transcript level (upper panel) using primers specific for the CysPc-C2L coding sequence in dek1Δloop and heterologous Loop replacement lines (At/Zm/MpLoop) after Cre/Lox removal of the resistance cassette. Lower panel, endogenous tubulin mRNA levels are shown as control. The primer’ sequences can be found in Supplemental Table S2.

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Supplemental Figure S6. Phyllid development failure in the dek1Δloop mutant. A, Young WT gametophore

with developing phyllids (arrowhead). B, (upper and lower panel) dek1Δloop gametophores with

protruding filamentous structures formed instead of the phyllids (arrows). Bar: 100 µm (A), 150 µm (B).

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Supplemental Figure S7. Sporophyte formation in WT, dek1Δloop and Loop complemented lines. A, WT

gametophore with mature sporophyte. B, dek1Δloop gametophore with no gametangia and no sporophyte.

C, PpLoop gametophore with maturing sporophyte. D, MpLoop gametophore with young sporophyte.

E, AtLoop gametophore with aborted gametangia and no sporophyte. F, ZmLoop line gametophore with

aborted gametangia and no sporophyte. Bar: 1 mm.

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Supplemental Figure S8. Micrographs of the Physcomitrella patens tissue used for RNA-seq analysis . A,

WT protonemata 6 days after inoculation. B, Δdek1 protonemata 6 days after inoculation. C, WT

protonemata with developing juvenile gametophores (arrowhead) 14 days after inoculation . D, Δdek1

protonemata with arrested buds (arrows) 14 days after inoculation. Bar: 200 µm.

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Supplemental Figure S9. Correlation between biological replicates. Pearson’s correlation coefficients for

the transformed FPKM values (log2(FPKM+1) indicated a high reproducibility within all conditions:

A, WT 6 days; B, WT 14 days, C, Δdek1 6 days, and D, Δdek1 14 days.

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Supplemental Figure S10. K-means clustering of the dataset. After filtering the data in order to keep only

informative genes (coefficient of variation larger then 0.5), k-means clustering was applied to find distinctive

groups of genes. In order to find the optimal number of clusters k, the average silhouette coefficient was

determined for a variety of ks. A. shows the silhouette coefficient obtained for k=4. B. Indication that this

was the optimal k among the tested range. C. Expression patterns of the four clusters divides the data into

four distinct groups corresponding the four experimental points.

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Supplemental Figure S11. Full dataset principal component analysis (PCA). Using DESeq2

(Anders and Huber, 2010), a principle component analysis based on the counts after variance

stabilizing transformation shows a clear separation between time points and moss lines.

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Supplemental Figure S12. Comparison of the dataset expressed genes with external datasets. For the 6 day

wild type data, we find a substantial overlap in expressed genes, when comparing them to other, external

data. Accessions for external data are GSM823365 and SRR072918.

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Supplemental Figure S13. Expression of PpDEK1 and control genes in the dataset. Expression levels for

a set of control genes, highlighting PpDEK1 deletion in the corresponding conditions. The height of the bars

corresponds to the reported FPKM, and the error bars represent the standard error (n=3).

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Supplemental Figure 14. Track view of PpDEK1 expression in the dataset. IGV visualization illustrates

the expression of DEK1 in various samples (-1, -2, -3 indicates the replicate number). In Δdek1, the ORF

signal is absent (6 days and 14 days). WT6-1unfilt shows the number of mapped reads before filtering for

uniquely mapped reads only. The light red SRR072918 track allows comparing the mapping observed in

our lines with external data. The last panel displays putative novel isoforms as reported by Cufflinks.

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Supplemental Figure S15. Transcriptome comparison between WT and Δdek1. GOSlim enrichment for

DEGs from interaction. For the genes reported as being significantly differently regulated between 6 days

and 14 days with respect to their genotype a GOSlim enrichment was performed. The color code corresponds

to FDR-adjusted p-values (white nodes: p-value >=0.05; others: p-value < 0.05).

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Supplemental Figure S16. Expression of selected PpVNS genes. For two (PpVNS1, PpVNS5) out of the

three tested PpVNS genes we found a significant difference in the expression levels between WT and Δdek1

at 14 days. For the remaining PpVNS genes no tests were performed due to the low abundance.

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Supplemental Table S1

Species AccessionAegilops tauschii EMT33050.1 a

Aquilegia coerulea AcoGoldSmith_v1.000031m.g b

Arabidopsis lyrata XP_002894501 a

Arabidopsis thaliana AT1G55350 b Brachypodium distachyon Bradi3g53020 b

Brassica rapa Bra037995 b

Camptotheca acuminata GenBank: GACF01058706.1 a

Cannabis sativa GenBank: JP475882.1 a

Capsella rubella Carubv10008068m b

Capsicum annuum GenBank: JW063188.1 a

Carica papaya evm.TU.supercontig_119.40 b

Ceratodon purpureus SRS140252 c

Chorispora bungeana KA022283.1 a

Cicer arietinum XP_004504206.1 a

Citrus clementina Ciclev10014012m.g b

Citrus sinensis orange1.1g000112m.g b

Costus pictus GenBank: JW231520.1 a

Cucumis sativus Cucsa.142290 b

Curcuma longa GenBank: JW811525.1 a

Eucalyptus grandis Egrandis_v1_0.000074m.g b

Fragaria vesca gene01602-v1.0-hybridGenlisea aurea EPS66151.1 a

Glycine max A Glyma08g13220 b

Glycine max B Glyma05g30080 b

Gossypium raimondii Gorai.003G153800 b

Hevea brasiliensis GenBank: JT914256.1 a

Hordeum vulgare ABW81402 a

Lactuca serriola GenBank: JO020465.1 a

Linum usitatissimum Lus10010313 b

Malus domestica_A MDP0000077683 b

Malus domestica_B MDP0000245785 b

Malus domestica_C MDP0000094595 b

Manihot esculenta cassava4.1_000045m.g b

Marchantia polymorpha A d

Marchantia polymorpha B d

Medicago truncatula Medtr8g088520.1Mimulus guttatus A mgv1a023650m.g b

Mimulus guttatus B mgv1a000044m.g b

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Musa acuminata GSMUA_Achr6P09310_001e

Nicotiana benthamiana AAQ55288 a

Oryza sativa AAL38190 a

Panicum virgatum Pavirv00022988m.g b

Phaseolus vulgaris Phvulv091008904m b

Physcomitrella patens XP_001774206 a

Populus trichocarpa A POPTR_0001s04110 b

Populus trichocarpa B POPTR_0003s20990 b

Prunus persica ppa000045m.g b

Ricinus communis XP_002523419 a

Selaginella moellendorffii A 235391b

Selaginella moellendorffii B 236021 b

Sesamum indicum GenBank: JP641107.1 a

Setaria italica SiPROV000042m.g b

Solanum lycopersicum Solyc12g100360. b

Sorghum bicolor XP_002468005 a

Thellungiella halophila Thhalv10011175m b

Theobroma cacao Thecc1EG038725 b

Thlaspi arvense GenBank: GAKE01002389.1 a

Utricularia gibba Scf00134.g10074.t1 f

Vitis vinifera XP_002285732 a

Zea mays NP_001105528 a

Nitella mirabilis This studyh

Klebsormidium flaccidum This studyh

Mougeotia scalaris This studyh

a NCBI GeneBank. b Phytozome. c The DEK1 sequence was retrieved from the SRS140252library deposit at NCBI GeneBank.Data were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/ in collaboration with the user community.d Sequences provided by Katsuyuki T. Yamato and Takayuki Kohchi (Liang et al., 2013). e http://banana-genome.cirad.fr/. f http://genomevolution.org/CoGe/. h Supplemental Table S5

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Supplemental Table S2. Primers used in plasmid construction, RT-PCR experiments, sequencing and for

making the Southern Blot probes.

Name of primer Purpose Primer sequence (5’ to 3’) delta loop fra-fw Genotyping

(Loss of Loop) AGACTCCAACCGCATCTCTTG

delta loop fra-rv Genotyping (Loss of Loop)

TCAATGCTGCTCCGTTGTAGC

delta loop diag fw Genotyping (Loop locus)

TAGCGACGTTGCCTAGTGCT

delta loop diag rv Genotyping (Loop locus)

ACAATGTGACGCTGCGCATC

V1/SP Cloning AGCCTTAGCTATAATCAGCAAC V1/ASP Cloning GAGCTTGATAGAAATTATACCCA At_Loop_ifc_SP Cloning GTGTCTGTTGTTAATCCATCAGCTGCAAGAAGAGATG At_Loop_ifc_ASP Cloning ACTGAAGAAGACCCATGCTCTCTCTGGTGTCCCC Zm_Loop_ifc_SP Cloning GTGTCTGTTGTTAATCCCTCAGTTGCAAGGATAGAC Zm_Loop_ifc_ASP Cloning ACTGAAGAAGACCCAAGCACGTATTGGAGATCCAG SP_Loop_Comp Cloning TGGGTCTTCTTCAGTGTGATC ASP_Loop_Comp Cloning ATTAACAACAGACACATGACGAAC Pp_Loop_Inverse_ASP Cloning TGGGTCTTCTTCAGTGTGATC Pp_Loop_Inverse_SP Cloning,

Sequencing TGGGTCTTCTTCAGTGTGATC

RT-Loop-F RT-PCR, Genotyping, Sequencing

TCCTGGTAGCTGGGTCCTACAG

RT-Loop-R RT-PCR, Genotyping, Sequencing

GCCAAGAGAAGTCCCACTCCAG

Loop-Genot-F Genotyping AGTGCATCCAAGTGGTCTCC Loop-Genot-R Genotyping TGACGCTGCGCAATCTATAAC Ter-F Genotyping TTCGCTCATGTGTTGAGCAT 35S-R Genotyping TCAATTGCCCTTTGGTCTTC PpL_5_Tar-Fw Sequencing TGACACAATCGGCCTTGCA PpL_3_Tar-Rv Sequencing TGACGTGTAAGGAGAGGTGGA Loop_Comp_Seq_F Sequencing GAGCAGTGTCAGATTGACAT Loop_Comp_Seq_R V1 Sequencing TCTTCCGTTGATGACAGTCACTGT 3TGS_Loop_seq_Rev Sequencing AGCGGCGAAAAAACGTAGAG PpL5_S_Sp Probe CCTAGTGCTTTGCTTTTCC PpL5_S_Asp Probe ACACTGCTCTACCAAATCT PpL3_S_Sp Probe GCACTTCTGCTGTCTCCT PpL3_S_Asp Probe AGACCTAACGCACCAATGA PpLL_S_Sp Probe TTGGGAGCTGTGGTAGTT PpLL_S_Asp Probe ACAGATAGCGGCGAAAAA G418-F Probe AGTTCATTTCATTTGGAGAGG G418-R Probe CTATGTCCTGATAGCGGT

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Supplemental Table S5. Overview of identified CysP transcripts from charophyte algae.

Species Transcript no. Domain structure Denotion*Klebsormidium flaccidum 1 Arm-CysPc-C2L T CysPcKlebsormidium flaccidum 2 TML-ArmKlebsormidium flaccidum 3 CysPc C CysPcMesostigma viride 1 Arm-CysPc-C2L T CysPcMesostigma viride 2 CysPc-C2L-C2L-C2L C CysPcMesostigma viride 3 TML-ArmMougeotia scalaris 1 CysPc-C2L T CysPcMougeotia scalaris 2 TML-ArmNitella mirabilis 1 TML-Arm-CysPc-C2L T CysPcNitella mirabilis 2 TMLNitella mirabilis 3 CysPc-C2L-C2 3 C CysPcNitella mirabilis 4 CysPc-CysPc-CysPc 4/1-4/3 C CysPcNitella mirabilis 5 CysPc-CysPc-CysPc-CysPc 5/1-5/4 C CysPcNitella mirabilis 6 CysPc 6 C CysPcNitella mirabilis 7 CysPc-CysPc 7/1-7/2 C CysPcSpyrogyra pratensis 1 LisH-CysPc-C2L C CysPcColeochaete orbicularis 1 TML-Arm-CysPc T CysPc* Denotion used in Figure 7. CysPc domains in bold are lacking the full complement of the catalytic triad

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Strain Bud initiation Bud development Gametophore development Gametangia Sporophyte References

WT 1 bud/15 cells filament

Proper division planes, stem cell activity on

Proper development, phyllids With differentiated marginal serrated cells and midrib, cca 30 phyllid blade cells/widest area

Present Present Perroud et al., 2014; this work

Δdek1 4 buds/15 cells filament

Aberrant cell division planes from the apical cell on, uneven cell wall, developmental arrest

Absent Absent Absent Perroud et al., 2014; this work

dek1Δloop 2 buds/15 cells filament

WT-like division in the bud apical cell, mersitematic activity on, progressive defects in bud patterning

Phyllid primordial cells initiated but blocked in further development - curved ~3 cell filaments formed instead of expanded phyllids

Absent Absent this work

2x35S:cPpDEK1 expressed from neutral locus in Δdek1 background

like WT WT-like division in the bud apical cell, mersitematic activity on; Phenotypical variations between strains in later bud patterning

Delayed development, phenotypical variations from WT-like gametophores to defective ones with no phyllid development

Present Absent Perroud et al., 2014

cPpCysPc-C2L in Δdek1 locus

like WT

WT-like division in the bud apical cell, stem cell activity on;

Delayed development, phenotypical variations from WT-like gametophores to stunted gametophores

Present Absent Perroud et al., 2014

cAtCysPc-C2L in Δdek1 locus

like Δdek1 like Δdek1 Absent Absent Absent Perroud et al., 2014

cZmCysPc-C2L in Δdek1 locus

like Δdek1 like Δdek1 Absent Absent Absent Perroud et al., 2014

MpLoop in locus

like WT like WT like WT Present Present (WT-like)

this work

AtLoop in locus

like WT like WT Gametophores are smaller with narrow lacerated phyllids – marginal serrated cells are not differentiated, ~4-8 phyllid blade cells/widest area, midrib often absent (when present, likely with defective organization)

Present Absent this work

ZmLoop in locus

like WT like WT Gametophores are smaller with narrow lacerated phyllids – marginal serrated cells are not differentiated, ~4-8 phyllid blade cells/widest area, , midrib often absent (present, with lower frequency than in AtLoop strain and likely with defective organization)

Present Absent this work

Supplemental Table S6. Overview of phenotypes of the dek1 mutants, DEK1 down-regulation and over-expression lines and genetic complementation experiments

in Physcomitrella patens and angiosperm species. A, Phenotypes of the Δdek1, DEK1 over-expression lines and genetic complementation experiments in Physcomitrella

patens. B, Phenotypes of the DEK1 over-expression and down-regulation lines and genetic complementation experiments in Arabidopsis thaliana. C, dek1 mutants,

DEK1 down-regulation and over-expression in maize, Arabidopsis, tobacco, and rice. A

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Complementation of the lethal dek1 mutants in Arabidopsis; over-expression and RNAi experiments. Constructs

Phenotypes References

Full length AtDEK1 CDS, genomic seq. under the native promoter

Fully rescued plants obtained Lid et al., 2005

Full length AtDEK1 cDNA tagged with GFP, under the pRPS5A

A range of phenotypes depending on the transgene expression: high expression needed for full complementation. Lines with low transgene expression show retarded growth, sterility; defective meristem functions; defective epidermis – lack of epidermal identity, enlarged epi. cells with no interdigitation, hyperproliferation

Johnson et al., 2008

AtDEK1 CysPc-C2L cDNA under the native promoter

Plants with an intermediate phenotype obtained – partial complementation with improved growth but severely defective aleurone and embryo

Tian et al., 2007

AtDEK1 CysPc-C2L cDNA tagged with GFP under the pRPS5A

Fully rescued plants with WT phenotype obtained. The lines with hight expression of the transgene showed more compact rossets with altered epidermis organization

Johnson et al., 2008; Liang et al., 2013

ZmDEK1 and PpDEK1 CysPc-C2L cDNA tagged with GFP under the pRPS5A, respectively

Fully rescued plants obtained in low frequency

Liang et al., 2013

MvDEK1 CysPc-C2L cDNA tagged with GFP under the pRPS5A, (Mesostigma viride)

No complementation Liang et al., 2013

AtDEK1 Arm-CysPc-C2L cDNA tagged with GFP under the pRPS5A

Fully rescued plants obtained; similar results as with pRPS5A:CysPc-C2L-GFP

Johnson et al., 2008

Constitutive expression of full length AtDEK1 genomic seq. under the 35S in WT background

Retarded flower growth, male sterility; distorted ovule integuments; disturbed dorsiventrality in leaves; irregular ities in petal and leaf epidermis patterning; altered cell morphology in subepidermal tissues (mesophyll ).

Lid et al., 2005

Constitutive expression of the AtDEK1 MEM genomic seq. under the p35S in WT background

A range of phenotypes – defective shoot apical meristem, no true leaves formation, lack of epidermal identity in cotypedons, defective epidermis organization, leaf radialization

Tian et al., 2007

Constitutive expression of the AtDEK1 MEMΔLoop genomic seq. under the p35S in WT background

WT phenotype (in Mikelsen`s Thesis, defective phenotypes observed, but with lower frequency than in AtDEK1-MEM-ox plants)

Tian et al., 2007

35S:RNAi Loss of epidermal identity; lack of organized meristem; in less severe knock-downs, radialized veg. organs (similar to AtDEK1-MEM-ox plants)

Johnson et al., 2005; Tian et al., 2007

B

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Species Null/strong alleles, endosperm

Null/strong alleles, embryo

Weak alleles, post-embryonal development

Down-regulation Over-expression Reference

maize No aleurone cells. Cell autonomous starchy endosperm /aleurone cell transdifferentiation (DEK1 gene on/off)

Embryo lethal, lack of the embryonic axis

Altered cell fate in leaf epidermis (ectopic bulbiform-like cells); altered cell morphology in subepidermal tissues; deformed plants

Becraft et al., 2002; Lid et al., 2002

Arabidopsis Failures in aleurone layer formation

Embryo lethality in pre-/globular stage; misoriented divisions in embryo proper and suspensor; lack of diff. protodermis (no embryonic L1 layer)

Lack of the giant cells in sepal epidermis (dek1-4)

Loss of epidermal identity; lack of organized meristem; in less severe knock-down mutants, radialized vegetative organs

Full genomic AtDEK1 under the 35S expression. Retarded flower growth, male sterility; distorted ovule integuments; disturbed dorsiventrality in leaves; irregularities in petal and leaf epidermis patterning; altered cell morphology in subepidermal tiisues

Johnson et al., 2005; Lid et al., 2005; Tian et al., 2007; Johnn et al, 2008; Roeder et al., 2012;

tobacco Hyperproliferation in leaf epidermal cells (lack of epidermal identy; lack of leaf lateral expansion; altered floral development (radialization); upregulation of the CycD/Rb genes

Ahn et al., 2004

rice Lack of aleurone cells on the ventral side of seeds

Embryo lethality at the globular stage

A range of phenotypes with altered embryo developemnt. Adaxialized rolled leaves with altered epidermal cell fate (ectopic bulbiform-like cells). Phenocopy of the mutants with altered splicing products (shifts in the MEM, ARM coding regions) and mutants with aa substitutions in the CysPc-C2L

Hibara et al., 2009

C

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Sheet1

Page 1

Supplemental Table S7 – Read mapping results

sample reads uniquely mapped reads % uniquely mappeddek_14-1 11688494 9908810 84,77 %dek_14-2 11964078 10226839 85,48 %dek_14-3 12244279 10361408 84,62 %dek_6-1 11961384 9915199 82,89 %dek_6-2 11588289 9551596 82,42 %dek_6-3 11422592 9526221 83,40 %wt_14-1 11458629 9579641 83,60 %wt_14-2 11554989 9741262 84,30 %wt_14-3 11879145 10106216 85,08 %wt_6-1 12744688 10420978 81,77 %wt_6-2 11835024 9782979 82,66 %wt_6-3 11718865 9765371 83,33 %

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Supplemental protocol 1: Transcriptome dataset validation

The four conditions presented distinct transcripts profiles with a high degree of

reproducibility between replicates (Supplemental Fig. S9). The four conditions present

four distinct profiles (see Supplemental Fig. S10). A principle component analysis

showed a clear separation between time points, probably reflecting the developmental

change, the gametophore growth (Supplemental Fig. S11). Overall, 20541 distinct

transcripts (Supplemental Table S3) were reported to be expressed (FPKM>1), a value in

line with other P. patens gametophytic similar dataset published (Xiao et al. 2011,

accession number GSM823365) or from publicly available NCBI-SAR similar P. patens

dataset (accession number SRR072918). For example, the 93 % of detected transcripts

are present in both WT6 day and SRR072918 (Supplemental Fig. S12). PpDEK1

transcript was present at low level at both time points in the WT compared to established

standard gene transcript accumulation (Supplemental Fig. S13, Lebail et al. 2013).

Finally confirming the clean PpDEK1 ORF deletion in the mutant, no reads for its ORF

could be found in Δdek1 as illustrated using Integrative Genomics Viewer

(Thorvaldsdottir et al., 2013) (Supplemental Fig. S14). Furthermore, DEK1 sequencing

read distribution along the gene structure confirmed the ORF splicing model as shown by

putative isoformes reported by cufflinks and cuffcompare (Supplemental Fig. S14).

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References:

Ahn JW, Kim M, Lim JH, Kim GT, Pai HS (2004) Phytocalpain controls the proliferation and differentiation fates of cells in plant organ development. Plant J 38: 969-981

Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome

Biol 11: R106

Becraft PW, Li K, Dey N, Asuncion-Crabb Y (2002) The maize dek1 gene functions in embryonic pattern formation and cell fate specification. Development 2002 129: 5217-5225

Hibara K, Obara M, Hayashida E, Abe M, Ishimaru T, Satoh H, Itoh J, Nagato Y (2009) The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice. Dev Biol 334: 345-354

Johnson KL, Degnan KA, Ross Walker J, Ingram GC (2005) AtDEK1 is essential for specification of embryonic epidermal cell fate. Plant J 44: 114-127 Johnson KL, Faulkner C, Jeffree CE, Ingram GC (2008) The phytocalpain defective kernel 1 is a novel Arabidopsis growth regulator whose activity is regulated by proteolytic processing. Plant Cell 20: 2619-2630 Le Bail A, Scholz S, Kost B (2013) Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes. PLoS ONE 8: e70998 Liang Z, Demko V, Wilson R, Johnson K, Ahmad R, Perroud P, Quatrano R, Zhao S, Shalchian-Tabrizi K, Otegui M, Olsen OA, Johansen W (2013) The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants. Plant J 75: 742-754 Lid SE, Gruis D, Jung R, Lorentzen JA, Ananiev E, Chamberlin M, Niu XM, Meeley R, Nichols S, Olsen OA (2002) The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc Nat Acad Sci USA 99: 5460-5465 Lid SE,Olsen L,Nestestog R,Aukerman M,Brown RC,Lemmon B,Mucha M,Opsahl-Sorteberg HG,Olsen OA (2005) Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally. Planta 221: 339-3515 Perroud PF, Demko V, Johansen W, Wilson RC, Olsen OA, Quatrano RS (2014) Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens. New Phytol 203: 794-804

Roeder AH, Cunha A, Ohno CK, Meyerowitz EM (2012) Cell cycle regulates cell type in the Arabidopsis sepal. Development 139: 4416-4427

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Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14: 178-92

Tian Q, Olsen L, Sun B, Lid SE, Brown RC, Lemmon BE, Fosnes K, Gruis DF, Opsahl-Sorteberg HG, Otegui MS, Olsen OA (2007) Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1. Plant Cell 19: 3127-3145 Xiao L, Wang H, Wan P, Kuang T, He Y (2011) Genome-widtrascriptome analysis of gametophyte development in Physcomitrella patens. BMC Plant Biol 11: 177