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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.
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).
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.
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.
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.
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.
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.
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.
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).
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.
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
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.
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
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
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
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
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
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)
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
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).
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
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