The DEK1 calpain Linker functions in three-dimensional body

1

Running title: Genetic analysis of DEK1 Linker function 1

2

Corresponding authors: 3

Wenche Johansen: [email protected] 4

Odd-Arne Olsen: [email protected] 5

6

The DEK1 calpain Linker functions in three-dimensional body patterning in 7

Physcomitrella patens 8

Wenche Johansen1§*, Ako Eugene Ako1§, Viktor Demko2, Pierre-François Perroud3, Stephan A. 9

Rensing3, Ahmed Khaleel Mekhlif1 and Odd-Arne Olsen2* 10

11

1)Hedmark University of Applied Sciences, P.O.Box 400, N-2418 Elverum, Norway. 12

2)Norwegian University of Life Sciences, P.O.Box 5003, N-1432 Ås, Norway. 13

3)Philipps University Marburg, Plant Cell Biology, Karl-von-Frisch-Str. 8, 35043 Marburg, 14

Germany. 15

§These authors contributed equally to the work. 16

*Corresponding authors. 17

18

Telephone numbers and e-mail addresses of the authors: 19

Wenche Johansen: +47 62517865 [email protected] 20

Ako Eugene Ako: +47 62517867 [email protected] 21

Viktor Demko: +47 96740783 [email protected] 22

Pierre-François Perroud: +49 64212822040 [email protected] 23

Ahmed Khaleel Mekhlif: +47 94140038 [email protected] 24

Plant Physiology Preview. Published on August 9, 2016, as DOI:10.1104/pp.16.00925

Copyright 2016 by the American Society of Plant Biologists

www.plantphysiol.orgon February 13, 2018 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

2

Stephan A. Rensing: +49 64212821940 [email protected] 25

Odd-Arne Olsen: +47 91581293 [email protected] 26

27

Footnotes: 28

Author contributions: 29

W.J. and O-A.O. conceived the research plans; W.J., V.D. and P-F.P. supervised the 30

experiments; A.E.A and W.J. performed most of the experiments; P-F.P., A.K.M and S.A.R 31

contributed to plant transformation and generation of mutants; V.D. contributed to phenotypic 32

characterization of the mutants; W.J. and A.E.A designed the experiments and analyzed the data; 33

W.J. and O-A.O. wrote the manuscript. 34

Financial sources: 35

This work was supported by research grant (FRIMEDBIO 240343) from the Norwegian 36

Research Council to O-A.O., the Norwegian University for Life Sciences (NMBU) for 37

personnel support and technical support at NMBU (P-F.P. and V.D.), and at Hedmark 38

University of Applied Sciences (HUAS) for personnel support (W.J.). Support to A.E.A was 39

from HUAS and the Norwegian Research Council. 40

41

One sentence summary: 42

Full and partial deletion of the DEK1 Linker implicate DEK1 in the three major developmental 43

phases of the moss Physcomitrella patens. 44

45

46



3

ABSTRACT 47

The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240 kDa key regulator of 48

three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The 49

activity of the cytosolic C-terminal calpain protease is regulated by the membrane anchored 50

DEK1 MEM, which is connected to the calpain via the 600 amino acid residue Linker. Similar to 51

the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the 52

similarity dropping sharply compared with orthologous charophyte sequences. Using 53

site-directed mutagenesis, we studied the effect on Physcomitrella patens development by 54

deleting the Linker and two conserved Linker motifs. Results show that removal of the Linker 55

has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the 56

conserved LG3 domain had a milder effect, perturbing leafy gametophore patterning and 57

archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. 58

patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker 59

sub-segment containing a potential calpain autolytic site severely disturbs gametophore 60

development. Finally, changing one of the three calpain active site amino acid residues results in 61

the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal 62

and DEK1 calpains, we propose that DEK1 MEM-Linker complex inactivates the calpain by 63

forcing the two calpain sub-units carrying the three amino acids of the active site apart. 64



4

INTRODUCTION 65

The three-dimensional architecture of land plant bodies are determined by the orientation 66

of cell walls deposited between dividing nuclei in developing organs. The ability to determine 67

cell wall orientation in various planes was a novel feature that evolved in the transition from 68

filamentous charophyte algae to the first land plants (Pires and Dolan, 2012). In contrast to land 69

plants, tip-expansion and fixed division planes of the cells drove a 2-dimensional (2D) growth 70

pattern of charophyte members represented by filamentous or discoid forms. The membrane 71

anchored DEFECTIVE KERNEL1 (DEK1) calpain is a candidate protein involved in 72

position-dependent cell wall deposition in the plant lineage (Perroud et al., 2014). Evidence for a 73

conserved function in land plants comes from the observation that DEK1 is essential for cell wall 74

orientation in land plants ranging from angiosperms to mosses, an evolutionary time span of 450 75

million years (Olsen et al., 2015). Support for a central function of DEK1 also comes from a 76

recent review where analyses of meristematic shoot tip transcriptomes of widely divergent extant 77

vascular plant lineage members suggests that different genes were recruited to regulate similar 78

meristematic processes during evolution. Exceptions identified include PIN-FORMED (PIN), 79

DEK1 and LONELY GUY1 (LOG1) (Frank et al., 2015) proteins that represent potential genetic 80

homologs in meristem function (Harrison, 2015). 81

The DEK1 calpain derives its name from the maize mutant dek1, which lacks aleurone 82

cells in the endosperm (Lid et al., 2002). The DEK1 protease core domain, CysPc, and the 83

additional C-terminal C2L domain are connected to DEK1 MEM, a 23 transmembrane (TM) 84

regulatory domain interrupted by a Loop segment via a Linker (previously referred to as Arm). 85

Phylogenetic analysis concluded that four ancestral calpain architectures existed approximately 86

1.5 billion years ago (Zhao et al., 2012). Of these, TML-calpains, to which DEK1 belongs, 87

consisted of a TM domain with at least 15 TM segments linked to the CysPc-C2L domains. The 88

common ancestor of the Chlorophyceae and Charophyceae algae is inferred to have contained 89

both cytosolic as well as TML-calpains (Demko et al., 2014). The Chlorophyceae group appears 90

to have lost TML-calpains, whereas the Charophyceae algae, the closest ancestors to land plants, 91

retained both cytosolic and TML-calpain variants. In the transition to land plants, cytosolic 92

calpains were lost, leaving DEK1 as the single calpain of land plants (Demko et al., 2014). We 93

have hypothesized that DEK1 evolved a novel function in the development of the 3-dimensional 94



5

(3D) architecture of land plants during the transition from charophyte land plant ancestors to land 95

plants (Demko et al., 2014). Our working hypothesis is that the calpain activity of DEK1 is 96

regulated by the MEM segment via an unknown ligand or stimulus or lack of such (release of 97

inhibition), and that the Linker transmits the signaling from MEM thereby activating the catalytic 98

calpain domain. The substrate(s) of the DEK1 calpain is unknown. 99

We are pursuing systematic genetic studies of the function and evolutionary conservation 100

of DEK1 and its component domains (Figure 1). In the first set of experiment, we focused on 101

CysPc-C2L by exploiting the observation that the Arabidopsis thaliana dek1-3 mutant phenotype 102

is complemented by A. thaliana CysPc-C2L under the control of the RIBOSOMAL PROTEIN5A 103

(RPS5A) promoter (Johnson et al., 2008). Results support the conserved nature of DEK1 function 104

in these experiments; the Physcomitrella patens DEK1 CysPc-C2L domains fully complements 105

the A. thaliana dek1-3 mutant phenotype (Liang et al., 2013). The CysPc domain of conventional 106

mammalian calpains (CAPN1 and CAPN2, also called µCL/calpain-1 and mCL/calpain-2, 107

respectively) consists of two sub-domains, PC1 and PC2, which are connected via a “hinge” 108

region; PC1 carries the active-site residue cysteine (Cys), whereas PC2 the two remaining 109

active-site residues histidine (His) and asparagine (Asn) (Strobl et al., 2000). The two 110

sub-domains also each harbor a conserved Ca2+ binding site (Moldoveanu et al., 2002). 111

Regulation of the CysPc domain of CAPN1 occurs via an electrostatic switch mechanism 112

operated via Ca2+ binding, causing the two CysPc sub-domains to swing into position around the 113

hinge, thus aligning the three active-site amino acid residues into an active configuration 114

(Moldoveanu et al., 2002). Additional levels of regulation of conventional calpains also exist that 115

depend on structures that are absent from DEK1. One example is calpastatin, a multi-headed 116

inhibitor that binds to the catalytic CysPc domain blocking access to the active site (Wendt et al., 117

2004). Three conserved features between DEK1 and animal CysPc suggest a similar activation 118

mechanism in plants and animals. First, the three active-site residues of the CysPc domain, as 119

well as their configuration with Cys located on PC1 and His and Asn located on PC2, are 120

conserved in DEK1 (Liang et al., 2013). Substituting the active site residue Cys with the amino 121

acid serine (Ser) inactivates the DEK1 calpain in vivo (Johnson et al., 2008) and in vitro (Wang 122

et al., 2003). Second, residues corresponding to the “hinge” are also conserved in the DEK1 123

CysPc domain (Wang et al., 2003; Liang et al., 2013), suggesting that a similar “swinging 124

motion” may be involved in activation of DEK1 calpain. Thirdly, the PC1 and PC2 amino acids 125



6

involved in Ca2+ binding in conventional mammalian CysPc domains (Moldoveanu et al., 2002) 126

are structurally and functional conserved in DEK1 (Liang et al., 2013). DEK1 CysPc harboring 127

substitution of these amino acids to alanine (Ala) fails to complement the A. thaliana dek1-3 128

mutant phenotype (Liang et al., 2013) suggesting that DEK1 calpain activity may be Ca2+ 129

dependent. This conclusion is potentially in conflict with the observation that recombinant 130

protein of maize CysPc-C2L displays a low, but significant in vitro proteolytic activity in the 131

presence of EDTA (Wang et al., 2003). Although sharing structural features, chimeric calpains 132

(the CysPc domain of animals fused to the C2L domain of DEK1) fail to complement the A. 133

thaliana dek1-3 mutant and co-evolution analysis also identified differences in the interactions 134

between the CysPc-C2L residues of DEK1 and classical calpains (Liang et al., 2013). 135

In the next series of studies of DEK1 function and evolution, we used the moss P. patens. 136

Moss represents one of, if not, the earliest extant relative of the first land plants, permitting 137

studies of ancestral DEK1 function. Second, mosses form a branched system of multicellular 138

filaments called protonemata, a gametophyte stage in which DEK1 is non-essential (Perroud et 139

al., 2014), allowing tissues to be cultivated and transformed, utilizing the potential of 140

homologous recombination for site-directed mutagenesis and gene replacement. This is in 141

contrast to other land plants, in which dek1 null mutations are lethal (Becraft et al., 2002; Lid et 142

al., 2005), severely restricting the ability to study DEK1 function. Third, DEK1 studies in 143

mosses are relevant for higher plants since its role in cell wall orientation is conserved in land 144

plants (Olsen et al., 2015). When P. patens spores germinate, the protonema, consisting of cells 145

extending by tip growth and dividing in one plane, represent a two-dimensional (2D) growth 146

form reminiscent of the charophyte algae predecessors of land plants. Shortly thereafter, buds are 147

initiated from the protonema, which develop into gametophores with phyllids (leaf-like 148

structures) and rhizoids (rooting structures), representing 3D organ forms typical of land plants. 149

At maturity, the gametophore produces archegonia and antheridia in a cluster at the apex of the 150

stem, which upon fertilization develop into diploid sporophytes containing the spores (Prigge 151

and Bezanilla, 2010). During its life cycle, P. patens develops several (eight) meristematic cells 152

that promote growth in the gametophyte and sporophyte generations (Kofuji and Hasebe, 2014). 153

In our first approach using P. patens to study DEK1 function, we delete the entire DEK1 154

gene (Perroud et al., 2014). In the resulting Δdek1 mutant, protonema growth appear normal, but 155



7

the mutant displays an over-budding phenotype, producing four times more buds than WT. Of 156

the four AP2-type transcription factors (APB1-4; for AINTEGUMENTA, PLETHORA and 157

BABY BOOM) shown to be indispensable for formation of the bud initial cell in P. patens 158

(Aoyama et al., 2012), APB2 and APB3 were found to be upregulated in the Δdek1 mutant 159

(Demko et al., 2014). In the Δdek1 mutant, abnormal cell divisions in the emerging bud result in 160

developmental arrest and the absence of gametophores. 161

Next, we investigated the function of the DEK1 Loop, a ~300 amino acid long segment 162

interrupting DEK1 MEM, by creating the Loop deletion mutant dek1Δloop. The dek1Δloop 163

mutant forms twice as many buds as WT (Demko et al., 2014). The division plane in the 164

dek1Δloop bud apical cell is perpendicular, similar to in WT, although with a slightly bended cell 165

wall. In contrast to the Δdek1 mutant, the dek1Δloop bud continues to divide, producing a 166

stem-like structure without emerging phyllids. Gametangia do not form. The liverwort 167

DEK1-Loop segment, sharing approximately 43% amino acid identity with the P. patens DEK1 168

Loop, is fully functional in the moss. However, the DEK1 Loop sequences from A. thaliana and 169

maize only partly complement the dek1Δloop phenotype; the numbers of buds, and the 170

positioning of the first division of the bud apical cell occur as in WT. However, gametophores 171

develop narrow phyllids and sporophyte is not formed (Demko et al., 2014). 172

In the present paper, we focus on the DEK1 Linker which we propose function to 173

transmit activation of DEK1 MEM by positional signaling into calpain activation. In this paper 174

we first show that the DEK1 Linker is highly conserved among land plants, but divergent from 175

orthologous charophyte algae sequences. Bioinformatic analyses identify functional elements, 176

including an LG3 domain and a potential autolysis site. To assess the importance of the Linker 177

segment, the LG3 domain and the putative autolysis site, we create partial P. patens DEK1 178

deletion mutants. We demonstrate the conserved nature of the LG3 domain between mosses and 179

liverworts by genetic complementation assay. Finally, we show that mutating one of the three 180

calpain active site amino acid residues generates the Δdek1 phenotype. We use these data to 181

discuss possible models for regulation of DEK1 calpain activation. 182

183



8

RESULTS 184

The land plant DEK1 Linker is highly conserved, contains a predicted β-sandwich fold and 185

a putative autolytic cleavage site. 186

The DEK1 Linker is ~600 amino acid long with a predicted molecular mass of 69 kDa 187

(Fig. 1A). Conserved domain database searches with DEK1 Linker as query identify a single 188

domain, Laminin_G3 (LG3) of ~165 amino acids, in the Linker segment at position 1443-1610 189

in the P. patens DEK1 sequence. Based on the identification of this domain, we divide the 190

Linker into the three sub-segments N, LG3 and C (Fig. 1A). Sequence alignment of 73 land plant 191

DEK1 Linker proteins and four orthologous sequences from charophyte algae (Supplemental 192

Table S1 and Supplemental Data S1) show that land plant DEK1 Linker sequences are highly 193

conserved (Figure 1B), with sequence identities ranging from 59-98% (Supplemental Table S2 194

and Supplemental Data S2). In contrast, the identity to the orthologous charophyte sequences is 195

moderate to low (<36%), and these are also highly divergent from each other (Supplemental 196

Table S2). Overall, sequence conservation between e.g. A. thaliana and P. patens is highest in 197

the N-segment (60% identity, 73% similarity), followed by the LG3 domain (55% identity and 198

68% similarity), and lowest in the C-segment (34% identity and 60% similarity) (Fig. 1B). In the 199

Linker N-segment, for which no conserved domain has been identified, several long stretches (up 200

to 16 amino acids) of 100% identity between the moss P. patens and angiosperm A. thaliana 201

sequences are present. For segment C, we identify one highly conserved stretch of 18 amino 202

acids, which are not present in motif databases. The position of this sequence coincides with a 203

proposed autolytic cleavage site for DEK1 (Johnson et al., 2008). 204

In BLAST searches, the land plant DEK1 LG3 domain align, although with low sequence 205

similarities (< 25 %) and insignificant E-values, to metazoan Sushi, von Willebrand factor type 206

A, EGF and pentraxin (PTX) domain-containing protein 1 and to prokaryotic Laminin G (LG) 207

domain-containing proteins, domains belonging to the Concanavalin A-like lectin/glucanases 208

superfamily (SCOPe v2.06; http://scop.berkeley.edu/). Protein domains of this superfamily adopt 209

a sandwich structure of 12-14 β-strands arranged in two sheets with a complex topology, referred 210

to as the legume lectin fold or β-sandwich fold. To investigate whether the LG3 domain of 211

DEK1 has the potential to adopt a similar fold, we predicted the structure of the P. patens DEK1 212

LG3 domain using three different threading servers, including I-TASSER, RaptorX and Phyre2. 213



9

All gave highly similar structures, all of which adopted a typical β-sandwich fold composed of 214

eight anti-parallel β-strands arranged in two sheets with loops connecting the β-strands on both 215

edges (Fig. 1C, I). All three servers modeled the fold with high confidence levels, for example an 216

I-TASSER C-score of -0.1 was obtained, indicating that the model was predicted with correct 217

global topology. In addition, average normalized Z-score of >2 shows good threading alignment 218

between template and the query sequence. Structure search of the model against RCSB PDB 219

(Research Collaboratory for Structural Bioinformatics Protein Data Bank; 220

http://www.rcsb.org/pdb/home/home.do) using PDBeFold (http://www.ebi.ac.uk/msd-srv/ssm/) 221

also gave similar top hits, belonging to the PTX family and the β-sandwich structure fold (Fig. 222

1C, II). 223

224

Deleting P. patens DEK1 Linker yields a phenotype similar to Δdek1 225

To assess the importance of the Linker for DEK1 function, we created a P. patens 226

dek1Δlinker mutant, in which a genomic sequence corresponding to 564 amino acids (aa) 227

(residues 1121-1684; XP_001774206.1) of the DEK1 Linker was removed (Fig. 2A). First we 228

transformed wild type (WT) protoplasts with the pBHRF-ΔLinkerG1 vector (Supplemental Fig. 229

S1A). This vector, containing 5’ and 3’ targeting sequences (TGSs) flanking the genomic region 230

encoding the DEK1 Linker, was designed to insert the Hygromycin phosphotransferase (hptII) 231

resistance cassette (HRC) flanked by loxP sites to intron 17. One of the resulting 232

hygromycin-resistant (HygR) lines, dek1ΔlinkerG1#20, gave PCR signals for targeted 233

integration, loss of DEK1 Linker and a single insertion event at the locus, as demonstrated by 234

Southern blotting analysis (Supplemental Fig. S1B). The dek1ΔlinkerG1#20 mutant displayed 235

the Δdek1 phenotype lacking gametophores (Perroud et al., 2014). Since insertion of the HRC 236

into an intron potentially can interfere with post-transcriptional processing of the DEK1 237

transcript, the HRC was removed by the Cre/lox mediated excision, resulting in the mutant 238

dek1ΔlinkerG1/cre. PCR genotyping and sequencing of the DEK1 locus from intron 12 to intron 239

26 confirmed removal of the HRC, leaving the loxP site in the fused intron between exons 17 and 240

24 of the DEK1ΔLinkerG1/cre locus. However, RT-PCR and subsequent sequencing of the 241

cDNA product revealed that the DEK1 transcript was not properly spliced, leaving the fused 242

intron in the processed mRNA creating a premature stop codon 5’ to the CysPc coding sequence 243



10

(Supplemental Fig. S1C). In order to remove the fused intron from the mutant, 244

dek1ΔlinkerG1/cre protoplasts were transformed with the pArrow-ΔLinkerG3 construct 245

(Supplemental Fig. S2A), containing the full exon 17 sequence fused in-frame with the 3’ of 246

exon 24, designed to insert the Neomycine phosphotransferase (nptII) resistance cassette (NRC) 247

after the DEK1 stop codon. One of the resulting mutants, dek1ΔlinkerG3#2, was subjected to 248

Cre/lox mediated excision to obtain the dek1Δlinker/cre#123 mutant line (hereafter referred to as 249

the dek1Δlinker mutant). The dek1Δlinker mutant was first characterized by PCR genotyping and 250

Southern blotting analysis (Supplemental Fig. S2B), confirming elimination of the DEK1 Linker 251

and showing no evidence of off-locus integrations. Secondly, RT-PCR using forward (Ex7-F) 252

and reverse (Ex30-R) primers annealing to DEK1 exons 7 and 30, respectively (Fig. 2A), showed 253

amplification of a single PCR product of expected sizes of 4.6 and 2.9 kbp for WT and 254

dek1Δlinker, respectively (Fig. 2B). Subsequent sequencing of the 2.9 kbp RT-PCR product 255

confirmed proper splicing of the truncated DEK1ΔLinker cDNA with exon 17 fused in-frame to 256

the 3’ end of exon 24 (Supplemental Fig. S2C). Quantitative RT-PCR was performed revealing 257

no significant changes in the steady-state DEK1 transcript level in 6 and 14 day old dek1Δlinker 258

mutant tissue compared to WT plants of the same age (Fig. 2C), supporting the conclusion that 259

deletion of the Linker did not affect the stability of the truncated DEK1 mRNA. 260

In the following section, we describe phenotypic characteristics of the dek1Δlinker 261

mutant compared to WT plants, a mutant lacking the entire DEK1 gene (Δdek1; Perroud et al., 262

2014) and a partial dek1 deletion mutant harboring a deletion within the DEK1 MEM segment 263

(dek1Δloop; Demko et al., 2014). The phenotypes of WT, Δdek1 and dek1Δloop plants are 264

shown in Fig. 3 A1-8, B1-8 and C1-8, respectively. In WT, one bud is typically initiated per 265

filament of 15 cells (Fig.3 A1). The bud initial cell divides by oblique division (Fig. 3 A2, 266

arrowheads) giving rise to the basal cell and the apical cell. The next division plane in the apical 267

cell is perpendicular to the first oblique division (Fig.3 A2, arrow). This division gives rise to the 268

bud apical initial (Fig. 3 A2, blue dot) and the first apical lateral cell (Fig. 3 A2, yellow dot). 269

Successive oriented divisions then lead to the formation of the gametophore apical cell 270

(tetrahedral stem cell) and primordial lateral domains (Fig. 3 A3). A WT young gametophore 271

with primordial phyllids is shown in Fig. 3 A4. Primordial phyllids are shaped by mitotic activity 272

of the phyllid apical cell (Fig. 3 A4, green dot) that divides by asymmetric oblique divisions. 273

Further mitotoc activity that takes place within the sectors of developing phyllid contributes to 274



11

phyllid lateral and longitudinal expansion. WT gametophores (Fig. 3 A5) form phyllids with 275

different morphologies depending on their position along the apico-basal axis of the 276

gametophore – a phenomenon known as heteroblasty. A typical WT phyllid from the middle part 277

of the gametophore is shown in Fig.3 A6. It has expanded lamina with pointed apex, serrulated 278

margins and central midrib. Archegonia, the female reproductive organs, are formed on 279

gametophore apices. During the final stages of their development, the neck apical cells become 280

vacuolated and swollen, and the apex opens (Fig. 3 A7). After fertilization of the egg cell 281

enclosed in the archegonium, a sporophyte is formed (Fig. 3 A8). 282

In contrast to WT, the Δdek1 mutant produces 4 buds per 15 cell filament (Fig. 3 B1) 283

while the dek1Δloop mutant produces 2 (Fig. 3 C1). The first oblique division of the bud initial 284

cell appears normal both in Δdek1 (Fig. 3 B2) and dek1Δloop (Fig. 3 C2). In Δdek1, the 285

division of the bud apical cell does not orient as in WT (Fig. 3 B2) and the cell divides in 286

variable random planes (Perroud et al., 2014). Moreover, the division of the resulting apical cell 287

is also misoriented while the apical lateral cell often does not continue to divide. Consequently, 288

bud development is arrested early after a few divisions (Fig. 3 B3). In dek1Δloop, the division 289

plane in the bud apical cell orients perpendicularly to the first oblique division, however the cell 290

wall tends to bend (Fig. 3 C2). As the bud continues to grow the pattern of cell divisions 291

becomes irregular (Fig. 3 C3). Phyllid initiation is abortive in dek1Δloop (Fig. 3 C4). In the 292

dek1Δloop mutant, expanded phyllids are never formed; instead, filamentous protrusions emerge 293

from a stem (Fig. 3 C5). 294

The dek1Δlinker mutant produces 2 buds per 15 cells filament compared to 1 bud in WT 295

and 4 buds in the Δdek1 mutant (Fig. 3 A-D1, Fig. 4). Similar to the Δdek1 mutant, the first 296

oblique division of the bud initial cell appears normal. However, the cell wall of the divided 297

apical cell points towards the lower ventral side of the bud and its intersection relative to the first 298

oblique division also vary compared to WT (Fig.3 D2). Resulting daughter cells then do not 299

expand as in WT and their successive divisions are misoriented which leads to disorganized buds 300

with abortive growth (Fig. 3 D3). In contrast to the Δdek1 mutant where buds are arrested early 301

after a few divisions, a proportion of dek1Δlinker buds continue to divide giving rise to larger 302

structures of variable sizes (Fig. 3 D4). No phyllid initiation was observed. From these results, 303

we infer that removal of the Linker segment leaves a minimal calpain activity sufficient to 304



12

support cell proliferation, although lacking organized division pattern. 305

Finally, in a control experiment, we re-targeted the DEK1 Linker sequence to the 306

DEK1ΔLinker locus as described in Supplemental Fig. S3A. PCR genotyping (Supplemental Fig. 307

S3B) and sequencing of the DEK1 cDNA (data not shown) confirmed correct insertion of the 308

Linker sequence. Two independent lines were obtained both forming normal gametophores with 309

fully expanded phyllids as shown for one of these lines (Supplemental Fig. S3C). 310

311

Deletion of the DEK1 LG3 domain perturbs leafy gametophore patterning and archegonia 312

development 313

We next studied the effect of deleting the LG3 domain in the dek1Δlg3 mutant. This 314

mutant was generated by transforming the pB-ΔLG3 construct (Supplemental Fig. S4A) into 315

dek1ΔlinkerG1#20 protoplasts, and harbors a deletion corresponding to 188 amino acids (aa 316

1426-1613; XP_001774206.1) that spans the LG3 domain (Fig. 2A). PCR genotyping and 317

Southern blotting analysis confirmed deletion of the LG3 sequence and no evidence of off-locus 318

integrations of the construct (Supplemental Fig. S4B). To verify in-frame removal of the LG3 319

coding sequence in the mutant, a 4.0 kbp RT-PCR product spanning from exon 7 to 30 was 320

amplified (Fig. 2B) and subsequently sequenced, confirming proper splicing of the truncated 321

DEK1 transcript with the 5’ end of exon 22 fused in-frame with the 3’ end of exon 23 322

(Supplemental Fig. S4C). No significant changes in the steady-state level of the DEK1 transcript 323

was observed between 6 and 14 day old WT and dek1Δlg3 tissue (Fig. 2C). 324

Removal of the DEK1 LG3 domain causes developmental changes in the gametophyte 325

that differ distinctly from Δdek1, dek1Δloop and dek1Δlinker mutants (Fig. 3). The bud initiation 326

rate is similar to WT (Fig. 3 A1 and E1, Fig. 4A). Early cell divisions in the dek1Δlg3 buds 327

follow the WT pattern. After the first oblique division of the bud initial, the division plane in the 328

apical cell orients perpendicularly (Fig. 3 E2) and the resultant daughter cells continue to divide 329

correctly to form tetrahedral stem cell (Fig. 3 E3, blue dot) and lateral segments of the young 330

gametophore (Fig. 3 E3). The effects of the LG3 deletion become apparent later during young 331

gametophore development. In particular, mitotic activity of the phyllid apical cell is reduced, 332

affecting phyllid primordia development (Fig. 3 E4). As a result, the dek1Δlg3 mutant forms 333



13

reduced gametophores with narrow phyllids lacking several features of the WT phyllids, 334

including the midrib (Fig. 3 E5 and E6). Archegonium development is also affected in the 335

mutant. The archegonium neck is deformed and does not open at the apex (Fig. 3 E7). 336

Consequently, fertilization is prevented and no sporophytes are formed in the mutant. 337

Altered phyllid and archegonia morphology in the dek1Δlg3 mutant is shown in more 338

details in Fig. 5. An example of heteroblastic phyllids typically seen in WT gametophores is 339

shown in Fig. 5A. Basal phyllids lack midrib and their marginal cells do not acquire highly 340

elongated pointed shape as the phyllids that develop later - also called upper phyllids. Mature 341

upper phyllids form a midrib that passes through from the base to the tip in the most expanded 342

phyllids. Proximal cells of the phyllid lamina are considerably larger than more distal cells. 343

Phyllid edges are formed of elongated pointed cells forming marginal serration. The juvenile 344

apical phyllids are also with serrulated edges and possess a midrib. In contrast to mature fully 345

expanded phyllids, the proximal cells in the apical phyllids are not yet elongated and continue to 346

divide. In the dek1Δlg3, basal phyllids consist of two or three files of cells with irregular shape 347

(Fig. 5B). Mature upper phyllids lack several characteristics of the WT, including a midrib, 348

marginal serration and acute tip. They do not expand as in WT and their narrow lamina lack the 349

typical proximo-distal distribution of cells with variable size. The same is true for the mutant 350

young apical phyllids. In WT, phyllids continue to expand by longitudinal and transverse cell 351

divisions that take place in sectors within the phyllid. From the phyllid morphologies in 352

dek1Δlg3, we infer that both phyllid apical cell activity and sectorial cell divisions within the 353

phyllid are impaired in the mutant. 354

Closer examination of the gametangia revealed that archegonium development was 355

affected in the dek1Δlg3 mutant. In WT, reproductive organ formation is highly organized as 356

described in detail by (Landberg et al., 2013). In the first stages of development, primordial 357

archegonium develops through a series of mostly anticlinal cell divisions forming a layer of outer 358

cells and inner cells (Fig. 5C). Upper inner cells elongate and give rise to the archegonium canal. 359

Basal inner cell divide asymmetrically and give rise to the egg cell. Upper outer cells also divide 360

anticlinaly and elongate to shape the archegonium neck. Basal outer cells divide both anti- and 361

periclinaly to form the basal part of archegonium with an egg cavity. During the final stages of 362

archegonium development, inner canal cells lyse, degrade and the resulting channel acquire 363



14

yellow-brown pigmentation (Fig. 5D). Content of the neck outer cells also degrade. The neck 364

apical cells become swollen and the apex is fully opened (Fig. 5D). In dek1Δlg3 mutant, the 365

development of archegonium appears irregular from the early stages with altered morphology 366

and organization of cells forming the neck (Fig. 5F). Defects become more apparent in the later 367

stages of the mutant archegonium development. The canal fails to form properly which is 368

marked by dislocated patchy brown pigmentation (Fig. 5G). The neck apical cells do not swell 369

and the apex fails to open. The egg and the egg cavity appear to form in the mutant (Fig. 5G). 370

These defects prevent fertilization and sporophyte is not formed in the mutant (Fig. 5H). No 371

major morphological defects in the mutant antheridia were detected (Fig. 5H), although we do 372

not exclude further anatomical/ultrastructural differences. 373

In our interpretation, the LG3 domain is needed for regulated activation of the calpain 374

during certain stages of gametophore development, including gametophore apical cell activity 375

and cell divisions in phyllids and archegonia. On the other hand, truncated DEK1 lacking the 376

LG3 domain is fully efficient to promote normal early bud and antheridia development. 377

378

379

The Marchantia polymorpha DEK1 LG3 domain complements the dek1Δlg3 mutant of P. 380

patens 381

To investigate whether DEK1 LG3 from angiosperms, liverwort and bryophytes are 382

functionally conserved, we introduced the Marchantia polymorpha (Mp) and A. thaliana (At) 383

DEK1 LG3 sequences into the DEK1 locus of the P. patens dek1Δlg3 mutant. First, to verify the 384

complementation assay, we retargeted the WT LG3 sequence into the dek1Δlg3 locus by 385

transforming dek1Δlg3 protoplasts with the pCR-PpLG3 construct (Supplemental Fig. S5A). 386

Three independent lines were obtained, all of which showed reversion to WT phenotype, 387

forming gametophores with fully expanded phyllids (Fig. 6). PCR genotyping confirmed 388

insertion of the WT LG3 sequence (Supplemental Fig. S5B). Next, we targeted the Mp DEK1 389

LG3 sequence to the dek1Δlg3 locus using the pCR-MpLG3 construct (Supplemental Fig. S5A). 390

Regenerating plants were screened by visual inspection, resulting in the identification of three 391

independent lines, all of which developed WT gametophore as shown for the line named MpLG3 392



15

in Fig. 6. PCR genotyping (Supplemental Fig. S5B) and sequencing of the heterologous DEK1 393

cDNA (data not shown) confirmed in-frame insertion of the Mp DEK1 LG3 sequence at the 394

DEK1 locus. These results show that the heterologous DEK1 LG3 domain from a liverwort, 395

sharing approximately 67% amino acid identity with the P. patens DEK1 LG3 domain, is 396

functional in moss gametophores, demonstrating functional conservation of DEK1 LG3 within 397

the groups of early diverging land plants. 398

Despite extensive testing, we were unable to identify regenerating plants exhibiting 399

gametophores with a different phenotype than that of dekΔlg3 after transforming mutant 400

protoplasts with pCR-AtLG3 (Supplemental Fig. S5A). This strongly suggests that the A. 401

thaliana DEK1 LG3 domain is not complementing the mutant phenotype or alternatively that no 402

targeted lines were obtained in this experiment. To explore this further, we chose an alternative 403

strategy using the construct pBHRF-AtLG3 designed to insert the A. thaliana LG3 sequence 404

together with HRC into the dek1Δlg3 locus as described in Supplemental Fig. S5C. 74 HygR 405

lines were obtained in this experiment and PCR genotyping was performed identifying one line 406

with proper 5’ and 3’ targeting signals. After cre/lox mediated removal of the HRC in this line, 407

the resulting PCR genotyping positive lines all displayed the Δdek1 phenotype. To investigate 408

DEK1 transcript processing, DEK1 cDNA was amplified from one of the after-cre lines and 409

subsequently sequenced, revealing that the A. thaliana DEK1 intron 21 was not spliced in the P. 410

patens DEK1AtLG3 transcript, thereby creating a premature STOP codon in the mRNA (data not 411

shown). More studies are therefore needed to firmly conclude whether DEK1 LG3 function is 412

conserved between mosses and angiosperms. 413

414

Deleting the potential autolysis site severely disturb gametophore development 415

We also created the dek1Δc-linker mutant, in which a genomic region corresponding to 416

71 amino acids (aa residues 1614-1684; XP_001774206.1) directly upstream of the DEK1 417

calpain domain was removed (Supplemental Fig. S6A). The rationale for making this mutant was 418

a previous study proposing an autolytic cleavage site in close proximity to the start of the A. 419

thaliana DEK1 calpain domain. The dek1Δc-linker mutant (Fig. 3) was verified using PCR 420

genotyping, Southern blotting analysis (Supplemental Fig. S6B), quantitative RT-PCR analysis 421



16

(Fig. 2C) and sequencing of the 4.0 kbp DEK1ΔC-Linker RT-PCR product (Fig. 2B and 422

Supplemental Fig. S6C). 423

The resulting mutant initiates approximately two buds per 15 cells filament (Fig. 3 F1, 424

Fig. 4). Early cell divisions of the dek1Δc-linker mutant bud showed similar pattern as the 425

dek1Δlinker mutant. The cell wall dividing the bud apical cell is leaning towards the lower 426

ventral side of the bud (Fig. 3 F2). The resulting cells then continue to divide in disorganized 427

manner forming distorted buds of various sizes (Fig. 3 F3). In contrast to Δdek1 and dek1Δlinker, 428

dek1Δc-linker bud cells continue to proliferate and form severely deformed gametophore-like 429

structures (Fig. 3 F4). In a number of cases, such gametophores form stems with filamentous 430

protrusions instead of phyllids similar to dek1Δloop (Fig. 3 F5). The remaining DEK1 calpain 431

activity is sufficient to enhance cell proliferation in the buds, however the structures lack 432

organized patterns and such residual activity is insufficient to initiate normal phyllid formation. 433

434

P. patens DEK1 function is dependent on the cysteine active site residue in the CysPc 435

protease core domain. 436

Finally, we created the P. patens dek10 single codon replacement mutant in which Cys1782 437

is replaced by a serine (Ser) (Supplemental Fig. S7A). Molecular analyses of the dek10 mutant 438

using PCR genotyping, Southern blotting analysis (Supplemental Fig. S7B), RT-PCR (Fig. 2B) 439

and subsequent sequencing of the DEK10 cDNA, showed targeted integration, no evidence of 440

off-locus integration and the Cys to Ser substitution, respectively. Quantitative RT-PCR analysis 441

confirmed that the steady-state level of the DEK10 transcript was comparable to the DEK1 442

transcript level in WT plants (Fig. 2C). 443

The dek10 mutant produces, on average, 3 buds per 15 cells filament, not significantly 444

different from the average bud number in the Δdek1 mutant (Fig. 3 G1, Fig. 4). The mutant does 445

not form leafy gametophores and bud development is affected at its early stage (Fig. 3), 446

mimicking the Δdek1 mutant phenotype (Perroud et al., 2014). After the first oblique division of 447

the bud initial, the apical cell fails to expand and divide as in WT (Fig. 3 G2). Bud growth stops 448

after few divisions without establishing the tetrahedral stem cell and lateral domains (Fig. 3 G3). 449

These results show first that an intact catalytic triad of the CysPc protease core domain is 450



17

essential for DEK1 function and that the Cys to Ser substitution gives the same effect as deleting 451

the DEK1 gene. The P. patens dek10 mutant phenotype thus confirms previous mutant studies in 452

A. thaliana (Johnson et al., 2008; Liang et al., 2013), and in vitro studies of maize DEK1 (Wang 453

et al., 2003) and animal calpains (Arthur et al., 1995) showing that calpain activity is dependent 454

on the active site residue cysteine. 455

456

DISCUSSION 457

The DEK1 protein of land plants form a separate phylogenetic clade in the streptophyte 458

lineage, with the highest degree of conservation in the calpain-specific catalytic domain CysPc, 459

showing more than 80% amino acid identity between the member species (Liang et al., 2013). As 460

shown here, replacement of the CysPc active site residue Cys with Ser in the P. patens dek10 461

mutant, generates the Δdek1 mutant phenotype (Fig. 3), confirming that an intact catalytic triad is 462

necessary for DEK1 activity in P. patens and underscores the calpain-dependent activity of 463

DEK1. A high degree of sequence conservation also exists for DEK1 MEM, with 54% overall 464

amino acid identity between A. thaliana and P. patens. Here we show that the high degree of 465

sequence conservation in the DEK1 land plant clade also extends to the ~600 amino acid residue 466

69 kDa Linker, the identity between land plant members ranging from 59 to 98%. The identity 467

between P. patens and A. thaliana is 60 %. Similar to DEK1 MEM and CysPc-C2L (Liang et al., 468

2013), DEK1 Linker sequence identity is dramatically reduced when comparing land plants to 469

charophytes, the algal predecessors of land plants, dropping to below 35%. The charophyte 470

sequences are also highly divergent from each other, suggesting a conserved functional role for 471

the DEK1 Linker in land plants that may differ from the function in charophyte algae. This 472

accords well with the proposal that a major change in DEK1 function occurred during the 473

evolutionary transition from charophyte algae to land plants (Demko et al., 2014). The phenotype 474

of the dek1Δlinker mutant confirms an essential role for the Linker in regulating DEK1 activity. 475

Notably, the effect of deleting the Linker is slightly less severe than deleting the entire DEK1 476

gene, suggesting that a DEK1 protein missing the Linker retains some residual calpain activity. If 477

so, this activity is sufficient to limit the increase in bud number to two buds per 15-cell filament, 478

compared to four in Δdek1, as well as to support cell proliferation beyond that of the Δdek1 479

mutant (Fig 3). 480



18

Within the Linker, the highest overall identity between P. patens and A. thaliana is in the 481

340 amino acid N-segment, 60%, including several stretches of up to 16 amino acids with 100 % 482

identity between P. patens and A. thaliana (Fig. 1B). In spite of the high amino acid sequence 483

conservation, no known structural element that may hint at the function of this part of the Linker 484

can be identified. 485

The identification of a conserved LG3 domain in DEK1 is supported by structural 486

predictions, the domain adopting a typical β-sandwich fold similar to the protein domains 487

belonging to the Concanavalin A-like lectin/glucanase superfamily of which LG3 is a member 488

(SCOPe). However, the number of predicted β-strands in DEK1 LG3 is lower, eight, compared 489

to 12-14 β-strands in other family members. The β-sandwich fold, also referred to as the lectin 490

fold, is a widely occurring fold present in 25 distinct protein families (SCOPe) of very diverse 491

functions. The predominant function of these proteins appears to be carbohydrate binding 492

(Chandra et al., 2001). However, despite the highly conserved structural fold, sequence similarity 493

among the different members is very low, identity in some cases being only 2% (Chandra et al., 494

2001), excluding prediction of structure-function relationships (Rudenko et al., 2001). The land 495

plant DEK1 LG3 domains display more than 59 % sequence identity and only insignificant 496

sequence similarities to other protein domains in the NCBI nr database, suggesting that DEK1 497

LG3 may represent a new family member of the Concanavalin A-like lectin/glucanase 498

superfamily. A number of the templates selected by the servers to build the models for DEK1 499

LG3 included PDB structures for PTX, e.g. serum amyloid P component (SAP) from Limulus 500

polyphemus and human C-reactive protein (CRP). Interestingly, these proteins are characterized 501

by Ca2+-dependent binding to their ligands, including carbohydrates, and function as soluble 502

pattern recognition molecules (Du Clos, 2013). Whether or not the DEK1 LG3 domain is binding 503

carbohydrates in a Ca2+-dependent process is yet to be determined. However, it is notable that 504

the DEK1 cysteine protease has been identified as a potential candidate involved in the initial 505

proteolysis and turnover of CELLULOSE SYNTHASE (CesA) in Gossypium hirsutum (cotton) 506

rosettes (Jacob-Wilk et al., 2006) responsible for cellulose biosynthesis in primary and secondary 507

cell walls (Festucci-Buselli et al., 2007). Both calcium signalling and CELLULOSE 508

SYNTHASE A have been implicated in moss bud development (Saunders and Hepler 1982; 509

Goss et al., 2012). Cytokinin treatment induces bud formation which coincides with elevated 510



19

intracellular Ca2+. Similar effect can be achieved after the treatment with a ionophore (Saunders 511

and Hepler 1982). We have previously shown that Δdek1 mutant responses to cytokinin-induced 512

bud formation (Perroud et al., 2014) and DEK1 is rather involved in a spatial control of bud 513

formation via negative regulation of APB transcription factors (Demko et al., 2014). 514

Disorganized buds are formed instead of leafy gametophores in P. patens cesa5 mutants (Goss et 515

al., 2012). Whether the DEK1 senses cell wall properties or is involved in the regulation of cell 516

wall remodeling needs to be further investigated. 517

Deletion of the LG3 domain in the P. patens dek1Δlg3 mutant is much less severe than 518

deletion of the Linker in dek1Δlinker, bud and leaf apical stem cell specification being unaffected 519

(Fig. 3). The effect of deleting the LG3 domain, however, does become apparent during later 520

developmental stages, as both phyllid and archegonia development are perturbed in the dek1Δlg3 521

mutant (Fig. 3), indicating that distinct DEK1 activities are required throughout 3D development 522

of the gametophyte. This observation suggests that the LG3 domain is involved in fine-tuning of 523

calpain activity and that the dek1Δlg3 mutant regulates calpain activity close to WT level, 524

sufficient to allow WT bud development, but insufficient to maintain WT phyllid and archegonia 525

patterning. Moreover, general reduction of the dek1Δlg3 gametophore growth indicates that the 526

activity of the gametophore stem cell is also reduced in the mutant. Thus, the DEK1 LG3 domain 527

may play its role depending on cellular context. Similar to what was demonstrated for the DEK1 528

Loop (Demko et al., 2014), the Marchantia polymorpha DEK1 LG3 domain complements the 529

dek1Δlg3 mutant of P. patens. The DEK1 LG3 domain from liverwort shares 67% amino acid 530

identity with the P. patens DEK1 LG3 domain, demonstrating functional conservation both in 531

MEM as well as in the Linker. Due to mis-splicing of the A. thaliana DEK1 transcript in the P. 532

patens dek1Atlg3 mutant we were not able to conclude whether or not DEK1 LG3 function is 533

conserved between angiosperms and mosses. 534

Compared to N and LG3, the 71 amino acid C-segment is the least conserved part of the 535

Linker, with an overall identity of 34% and a similarity of 60% between land plants (Fig. 1B). 536

Interestingly, a highly conserved stretch of 18 amino acid residues is located ~36 amino acids 537

upstream of CysPc, corresponding approximately to the location of a proposed autolytic cleavage 538

site of the DEK1 calpain (Johnson et al., 2008). The consensus sequence of this amino acid 539

stretch is YDRDDVDWDGQYSSGRKR, the residues underlined being 100% conserved in land 540



20

plant DEK1 proteins included in this study. The consensus sequence is not recognized as a 541

conserved motif in current databases. The signal for animal calpain cleavage and the structural 542

clues for substrate recognition are largely unknown (Sorimachi et al., 2012). However, in a 543

recent study, both primary and higher order structure were identified as determinants for CAPN1 544

and CAPN2 substrate specificity (Shinkai-Ouchi et al., 2016). In an effort to determine the 545

amino acid preference around the cleavage site Tompa et al. (2004) analyzed 106 CAPN1 and 546

CAPN2 cleavage sites in 49 substrates compiled from the literature. The preferred residues in the 547

P2 position were identified as Leu, Val and Thr, and in P1, Tyr, Arg and Lys. Small hydrophilic 548

residues, Ser and to a lesser extent Thr and Ala were found to occur more often in P1’. In 549

addition, a high preference for Pro in position P3 and P2’-P4’, was identified. This sequence 550

pattern is not present in the consensus sequence of the conserved 18 amino acid residue 551

C-segment site. Thus, if this sequence is indeed a DEK1 autolytic cleavage site, the DEK1 552

calpain has different cleavage site preferences than conventional animal CAPN1 and CAPN2 553

proteins. 554

The functional significance of the Linker C-region is clearly demonstrated by the 555

phenotype of the dek1Δc-linker mutant, resembling dek1Δlinker in severity during early stages of 556

bud development (Fig. 3). In both mutants, the tetrahedral apical cell is not formed; however, 557

mitotic activity is present allowing cell proliferation beyond that of the Δdek1 mutant, resulting 558

in buds of variable sizes. In contrast to the dek1Δlinker, dek1Δc-linker proliferates to form 559

stem-like structures, similar to dek1Δloop (Demko et al., 2014) (Fig. 3). These observations 560

suggest that the dek1Δc-linker and dek1Δloop mutants regulate DEK1 calpain activity to similar 561

levels. Further analyses are required to determine whether the conserved 18 amino acid residue 562

site alone causes the dek1Δc-linker phenotype. 563

The multiple phenotypes obtained when deleting different regions of DEK1 point to 564

different functions of the different parts of the Linker throughout P. patens development. A fuller 565

understanding of DEK1 function has to await the 3D structure of DEK1. Meanwhile, our 566

working hypothesis for DEK1 function is that the DEK1 MEM and Linker segments regulate the 567

activity of CysPc-C2L. The observation that deletion of the Loop leads to a severe phenotype 568

(Demko et al., 2014; Fig. 3) supports the role of DEK1 MEM in regulating DEK1 calpain 569

activity. The exact role of the Loop in positional sensing, if any, needs further studies. Upon 570

activation of DEK1 MEM our working model for DEK1 regulation proposes that a 571



21

conformational change of DEK1 MEM activates the CysPc-C2L moiety in a process that 572

involves the Linker. The activated protease then cleaves the Linker and is thereby released in its 573

active form ready to cleave its unknown substrate(s). Although the mode of activation is 574

unknown, the structural similarities between DEK1 CysPc and CAPN2 CysPc make it tempting 575

to speculate that DEK1 calpain activation occur by a similar mechanism. If so, a change in the 576

conformation of the activated MEM-Linker allows the CysPc subdomains PC1 and PC2, to 577

swing close together, thereby forming an active site. In CAPN2, the inactive form represents the 578

ground state of the protein in which electrostatic forces in an acidic loop within C2L is partly 579

responsible for separation of PC1 and PC2 (Strobl et al., 2000). Activation by Ca2+ occurs in two 580

steps, first by releasing constraints imposed by the circular arrangement of the domains, then by 581

cooperative binding of Ca2+ to PC1 and PC2 (Khorchid and Ikura, 2002). We propose that the 582

DEK1 MEM-Linker acts via a similar mechanism, although it remains to be determined whether 583

or not the ground state of the DEK1 calpain is an active state or not. It is possible that both 584

endogenous DEK1 as well as ectopically expressed CysPc-C2L that is capable of complementing 585

the A. thaliana dek1-3 mutant phenotype all require binding of Ca2+ to be active. Indeed, the 586

observation that disruption of the conserved amino acids coordinating Ca2+ binding sites in 587

conventional calpain makes the A. thaliana CysPc-C2L incapable of complementing the dek1-3 588

mutant phenotype (Liang et al., 2013) supports this interpretation. The observation that deletion 589

of the Linker does not lead to DEK1 activation suggests that the Linker alone does not function 590

to inactivate CysPc, but rather that MEM-Linker acts together. This conclusion is also supported 591

by the observation that ectopically expressed Linker-CysPc-C2L is capable of complementing 592

the A. thaliana dek1-3 mutant phenotype (Johnson et al., 2008). The structure of DEK1 593

MEM-Linker is unknown. However, it is interesting to note that part of the MEM shows 594

similarity to members of the major facilitator superfamily (MFS) of proteins of bacterial origin 595

(Demko et al., 2014). Members of this family have evolved from transporters, via dual function 596

molecules referred to as transceptors to receptors (Yan, 2013). Given the long evolutionary 597

history of TML-calpains and the DEK1 clade of TML-calpains, it is an interesting possibility 598

that a similar functional change took place with DEK1. 599

600

CONCLUSION 601



22

In contrast to higher plants, where loss of function alleles of DEK1 are lethal, the protonemata 602

stage of mosses are not critically affected by loss of DEK1. Interestingly, the unique moss 603

protonemata stage represents an image of the transition from charophyte algae to land plants. 604

Initially, it grows in a 2D mode typical of algae predecessors of land plants. It then switches to 605

the 3D growth form of land plants, initially in the form of the gametophore bud. DEK1 plays a 606

role in two major processes related to 3D growth; first by affecting the number of buds via APB 607

transcription factors. Secondly, by its essential function in positioning of the cell wall resulting 608

from the first division of the apical bud cell. The present study completes a series of 609

investigations into the functional effect of deleting specific domains of DEK1, including the 610

entire gene and the DEK1 Loop. Here we first show that the Linker performs an essential 611

function in the regulation of the DEK1 calpain activity; loss of the Linker has nearly the same 612

effect as loss of the entire DEK1 gene. In addition, the current analysis also identifies an 613

important function in the Linker C-segment containing a highly conserved 18 amino acid long 614

sequence, the deletion of which also has a severe effect on moss development. One potential 615

function of this sequence is to serve as substrate for the DEK1 calpain protease, although the 616

sequence does not represent a typical target sequence for conventional mammalian calpains. The 617

second conserved Linker sub-sequence is LG3, the deletion of which leads to less dramatic 618

perturbation of moss development. Earlier studies showed that the DEK1 Loop sequence of 619

Marchantia polymorpha is functional in moss, but sequences from angiosperm are not. 620

Similarly, the LG3 sequence of M. polymorpha functions in moss, but not angiosperms LG3 621

sequences. Earlier studies have shown the essential nature of the cysteine residue of the active 622

site of CysPc. Here we confirm the essential nature of the P. patens Cys1782 residue to DEK1 623

function. Finally, we propose that DEK1 MEM-Linker in an inactive state inhibits the calpain 624

activity by forcing apart the CysPc PC1 and PC2 subdomains. In an active state, this inhibition is 625

abolished and the two subunits swing closer bringing the three active site amino acid residues 626

together into its active site configuration. 627



23

MATERIAL AND METHODS 628

Plant material and growth conditions 629

The Physcomitrella patens Gransden strain was used throughout this study. Tissue maintenance 630

and production was performed on BCDA medium as described by Cove et al. (2009). P. patens 631

tissue and protoplasts were grown under long day conditions (16 hours light [70 to 80 632

µmolm-2s-1]/8 hours dark) at 25 °C. Growth medium was supplemented with 20 µgl-1 of 633

Hygromycin B or 30 µgl-1 of G418 for selection of transformed cells. Tissue for phenotypic 634

characterizations was grown on BCD medium unless otherwise stated. Cultures for bud counting 635

were established as follows: BCD-containing Petri dish was inoculated with 16 equally spaced 636

spot inoculums consisting of 10 µl of protonemal tissue suspension. After 14 days of growth, 637

buds were counted on one hundred filaments using a dissecting microscope, and the average 638

number of buds per filament calculated. Buds were counted on 15 cells from the tip of the 639

filaments. The one hundred bud-containing filaments were randomly chosen from different areas 640

of the plate. Tissue for sporophyte production was grown on sterile Jiffy7 soil blocks placed in 641

glass jars under short day conditions (8 hours light [70 to 80 µ molm-2s-1]/16 hours dark) at 15 °C 642

and manipulated as described (Perroud et al., 2011). Tissue for Real-time Quantitative Reverse 643

Transcription PCR was grown and harvested as follows: protonemal tissue grown on BCDA 644

under long day conditions was collected, homogenized in sterile water and inoculated on BCD 645

medium overlaid with cellophane discs. The tissue was collected after 6 and 14 days of growth, 646

snap-frozen in liquid nitrogen and stored at -80 °C until further processing. 647

648

In silico analyses 649

The orthologous DEK1 sequences used in this study are listed in Supplemental Table S1. 650

To identify and extract the amino acid (aa) sequences corresponding to the DEK1 Linker, 651

defined as the segment between the transmembrane spanning domain (MEM) and the CysPc 652

domain (Fig. 1A), full-length DEK1 sequences were submitted to the Simple Modular 653

Architecture Research Tool (SMART) server (http://smart.embl-heidelberg.de). The extracted 654

DEK1 Linker sequences were further submitted to Pfam (pfam.xfam.org) and the Conserved 655

Domain Database (CDD) (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to identify putative 656



24

conserved domains. Pfam domain models were extracted using SMART. Multiple sequence 657

alignment (MSA) of DEK1 Linker sequences were performed with the L-INS-I algorithm using 658

MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley, 2013). 659

Pairwise comparison of each pair of sequences in the MSA was calculated as the percentage of 660

identical residues in alignment positions between the two sequences using the software CLC 661

Genomic Workbench v7. For predicting the three-dimensional structure of the P. patens DEK1 662

LG3 domain, the aa sequence corresponding to LG3 (residues 1426-1613; XP_001774206.1) 663

was submitted to three different fold recognition web servers, including RaptorX 664

(http://raptorx.uchicago.edu/), I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) 665

and Phyre2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). The SAVES 666

metaserver (http://services.mbi.ucla.edu/SAVES/) was used for structural analysis and validation 667

of the generated models to verify their stereochemical quality and overall structural geometry. 668

669

Construction of plasmids for gene targeting 670

Oligonucleotides used for vector constructions are listed in Supplemental Table S3. The 671

schematics for the gene deletion and knock-in complementation constructs are shown in 672

Supplemental Figs. S1-S7. Plasmids were generated using the In-Fusion® HD Cloning kit 673

(Clontech Laboratories) according to the manufacturer’s instructions, unless otherwise stated. 674

The PCR generated fragments used in the In-Fusion reactions were amplified using Thermo 675

Scientific® Phusion High-Fidelity DNA Polymerase. To verify the constructed vectors, 676

restriction-digestion and DNA sequencing analyses were performed. Nucleotide (nt) numberings 677

are relative to the A1TG start site of the P. patens (Pp) DEK1 sequence (Pp3c17_17550; 678

www.phytozome.net). 679

Two plasmid vectors, named pBHRF-ΔlinkerG1 and pArrow-ΔlinkerG3 were used to 680

generate the Pp dek1Δlinker mutant. GenScript Inc. (USA) performed fragment synthesis and 681

cloning of the pBHRF-ΔlinkerG1 vector. In short, the 5’ targeting sequence (TGS), Pp DEK1 nt 682

7249-8346 flanked by 5’ HindIII and 3’ NruI recognition sequences, and the 3’ TGS, Pp DEK1 683

nt 11545-12546 flanked by 5’ SpeI and 3’ NsiI recognition sequences were synthetized de novo. 684

The 5’ and 3’ TGS were cloned sequentially into pBHRF (Schaefer et al., 2010) using 685

HindIII/NruI and SpeI/NsiI restriction enzyme pairs, respectively, creating construct 686



25

pBHRF-ΔLinkerG1 (Supplemental Fig. S1A). Prior to Pp transformation, pBHRF-ΔlinkerG1 was 687

digested with BmrI and PacI. The pTDek1-3' plasmid (Perroud et al., 2014), which contains a 3’ 688

TGS of 1003 bp 3’ to the DEK1 termination code, was used to build the pArrow-ΔlinkerG3 689

construct. The 5’ TGS of the latter construct, designed to contain a mutagenized Pp DEK1 690

fragment spanning from nt 7250-14569 with a 3398 bp deletion from nt 8321-11718, was 691

amplified using two PCR-generated fragments (fragment A and B) in an overlapping PCR 692

reaction. Fragments A and B were first separately PCR-amplified using primer pairs SP-A and 693

ASP-A, and SP-B and ASP-B, respectively with genomic Pp DNA as template. The 5’ TGS was 694

then amplified with primer set SP-A and ASP-B in a second round of PCR using fragment A and 695

B as template. The SP-A and ASP-B primers were designed to add 15 bp 5’ and 3’ extensions to 696

the resulting PCR product facilitating insertion of the 5’ TGS into SmaI digested pTDek1-3' 697

using In-Fusion reaction. The resulting construct, pArrow-ΔLinkerG3 (Supplemental Fig. S2A) 698

was digested with BamHI prior to Pp protoplast transformation. 699

In order to generate the P. patens dek1Δlg3 mutant, construct pB-ΔLG3 was used. This 700

plasmid, containing a mutagenized Pp DEK1 fragment spanning from nt 7249 to 12546 701

harboring a 673 bp deletion from nt 10567 to 11239 spanning the DEK1 LG3 sequence, was 702

made in several steps; first we PCR-amplified an In-Fusion fragment containing Pp DEK1 nt 703

8347-11544 with primers SP_PpLinker and ASP_PpLinker, and genomic Pp DNA as template. 704

This insert was mixed with linearized vector pBHRF-ΔlinkerG1, produced by PCR using primers 705

SP_Inf1 and ASP_Inf1 to exclude the sequence between the 5’ and 3’ TGSs in this vector (e.g. 706

the HRC). The insert and linearized vector were ligated in an In-Fusion reaction, resulting in 707

plasmid pB_Linker, containing a full Pp DEK1 fragment spanning from nt 7249 to 12546. 708

Finally, a second PCR amplification was performed to delete nt 10567-11239, using pB_Linker 709

as template, and primers SP_Inf2 and ASP_Inf2, resulting in the generation of the pB-ΔLG3 710

construct (Supplemental Fig. 4A). The construct was digested with RsrII and PacI prior to Pp 711

protoplast transformation. 712

To generate the P. patens dek1Δc-linker mutant, the construct pBHRF-ΔC-2G was used. 713

The 3’ TGS of this construct, spanning from nt 8402 to 12893, contains a 479 bp deletion (nt 714

11240-11718) corresponding to the Linker C-segment (Fig. 1A). Construct pBHRF-ΔC-2G was 715

built from the pBHRF-ΔlinkerG2 plasmid. The latter plasmid was generated by GenScript Inc. 716

(USA) as follows: In short, the 5’ TGS, Pp DEK1 nt 7249-8404, flanked by 5’ HindIII and 3’ 717



26

NruI recognition sequences, and a mutagenized 3’ TGS, PpDEK1 nt 8402-12893 flanked by 5’ 718

SpeI and 3’ NsiI recognition sequences, were synthetized de novo. The 3’ TGS was designed to 719

contain the following sequence (from 5’ to 3’): 3’ end of intron 17 to the 5’ end of exon 18 (nt 720

8402-8461) fusion in frame with the 3’ end of exon 24 to the start of intron 26 (nt 11719-12893). 721

The 5’ and 3’ TGS were cloned sequentially into pBHRF (Schaefer et al., 2010) using the 722

HindIII/NruI and SpeI/NsiI restriction enzyme pairs, respectively, creating plasmid 723

pBHRF-ΔlinkerG2. We then PCR amplified, from genomic Pp DNA, an In-Fusion fragment 724

containing Pp DEK1 nt 8462-11718 with primers SP_2G_E and ASP_2G_E, designed to add 15 725

bp 5’ and 3’ extensions complementary to the ends of the linearized vector. The insert was mixed 726

with linearized (at Pp DEK1 nt position 8461) pBHRF-ΔlinkerG2 DNA, produced by PCR using 727

primers SP_2G_F and ASP_2G_F, then ligation using an In-Fusion reaction was performed, 728

resulting in plasmid pBHR-LinkerG2. Finally, a second PCR amplification was performed to 729

delete nt 11240-11718, using pBHR-LinkerG2, and primers SP_2GDC_Inf and ASP_2GDC_Inf, 730

followed by an In-Fusion reaction, resulting in the generation of the pBHRF-ΔC-2G construct 731

(Supplemental Fig. S6A). The construct was digested with BmrI and PacI prior to Pp protoplast 732

transformation. 733

734

To generate the P. patens dek10 mutant, the construct pArrow-DEK10 was used. The 5’ 735

TGS in this construct contains a mutagenized PpDEK1 fragment spanning from nt 11763 to 736

14569 containing the two substitutions G to C and C to T at nt positions 1186 and 1187, 737

respectively. The WT PpDEK1 fragment (nt 11763-14569) was first amplified from genomic Pp 738

DNA using primers SP-PpCysPc and ASP-PpC2L designed to insert 5’ SmaI and 3’ BsiWI 739

recognition sequences, and then cloned into the Zero Blunt PCR cloning vector (Invitrogen, 740

Carlsbaden, USA), resulting in plasmid pCR-PpCalp. To introduce the two single point 741

mutations, the pCR-PpCalp plasmid was subjected to mutagenesis using GeneTailor™ 742

Site-Directed Mutagenesis System (Invitrogen) and the mutagenic primer pair SP-PpCysMut0 743

and ASP-PpCysMut0. The mutagenized SmaI/BsiWI restriction fragment of pCR-PpCalp 744

fragment was finally sub-cloned into SmaI/BsiWI digested pTDek1-3', resulting in construct 745

pArrow-DEK10 (Supplemental Fig. S7A). The construct was digested with SmaI and PacI prior 746

to transformation into P. patens. 747

748



27

Complementation constructs were assembled to re-insert the Linker and LG3 sequences 749

into the deleted loci. Constructs pCR-PpLinker and pCR-PpLG3 were used to re-insert the P. 750

patens DEK1 Linker and DEK1 LG3 sequences into their native loci, respectively. In short, a 751

DNA fragment (5263 bp) spanning the Linker in addition to 5’ and 3’ flanking regions (nt 752

7368-12630) was PCR amplified from genomic Pp DNA using primers Linker5’-Fw and In25-R. 753

Similar, a DNA fragment (3357 bp) spanning LG3 and 5’ and 3’ flanking regions (nt 754

9274-12630) was PCR amplified using primers ArmSeq3 and In25-R. The resulting fragments 755

were each cloned into the Zero Blunt PCR cloning vector, giving plasmid pCR-PpLinker 756

(Supplemental Fig. S3) and pCR-PpLG3 (Supplemental Fig. S5A), respectively. We also built 757

two vectors aimed to test heterologous LG3 sequences from A. thaliana (At) and Marchantia 758

polymorpha (Mp). In these constructs, chimeric LG3 sequence from At (nt 6741-7450; relative to 759

the ATG start site; AT1G55350) and Mp (nt 1258573-1259287; scaffold 26, JGI scaffold 760

genomic data v3.1) flanked by P. patens DEK1 5’ and 3’ targeting sequence were constructed as 761

follow: To generate the At and Mp Linker In-Fusion inserts, forward and reverse gene specific 762

primers containing 5’ and 3’ 15 bp extensions complementary to the flanking regions of the P. 763

patens LG3 sequence in the pCR-PpLG3 plasmid was used to PCR amplify At (primers 764

SP-AtLG3-Inf and ASP-AtLG3-Inf) and Mp (primers SP-MpLG3-Inf and ASP-MpLG3-Inf) from 765

genomic DNA. These inserts were each mixed with linearized vector pCR-PpLG3, produced by 766

PCR amplification using primers SP_Inf2 and ASP_Inf2x to exclude the Pp LG3 sequence. The 767

inserts and linearized vector were ligated using In-Fusion Cloning reactions, resulting in 768

constructs pCR-AtLG3 and pCR-MpLG3 (Supplemental Fig. S5A). The complementation 769

constructs were digested with EcoRI prior to P. patens protoplast transformation. An additional 770

vector, pBHRF-AtLG3, was constructed to target the At LG3 sequence together with HRC into 771

the dek1Δlg3 locus. First, we PCR amplified an In-Fusion fragment containing Pp DEK1 nt 772

8347-11544 with primers SP_Inf_6N and ASP_Inf_6N and genomic Pp DNA as template. This 773

insert was mixed with linearized vector pBHRF-ΔlinkerG1, produced by PCR using primers 774

SP_Inf_5N and ASP_Inf_5N. The insert and linearized vector were ligated in an In-Fusion 775

reaction resulting in plasmid pBHRF-Linker, containing the Pp DEK1 nt 7249-11544 as the 776

5’TGS. The At LG3 sequence fragment (nt 6741-7450; relative to the ATG start site; 777

AT1G55350) was amplified from genomic At DNA using primers SP-AtLG3_Inf and 778

ASP-LG3_Inf. Vector pBHRF-Linker was PCR-amplified to delete the Pp LG3 sequence using 779



28

primers SP_Inf2 and ASP_Inf2X. Finally, the linearized vector and the AtLG3 amplicon were 780

ligated in an In-Fusion reaction creating vector pBHRF-AtLG3 (Supplemental S5B). 781

782

P. patens transformation procedure 783

P. patens protoplast production and transformation was performed according to Schaefer 784

and Zrÿd (1997) as modified by Cove et al. (2009) with 15 µg of linearized plasmid DNA used 785

per transformation. Briefly, transformed protoplast regeneration and selection was performed by 786

transferring the tissue to different media according to the following sequence: 7 days protoplast 787

regeneration on PRMB medium, 7 days of selection on BCDA medium supplemented with the 788

appropriate antibiotic, 14 days of growth on BCDA medium and 7 days on BCDA supplemented 789

with the appropriate antibiotic. Resistant plants were individually transferred to fresh BCDA 790

medium and used for genotyping after sufficient growth. 791

Cre recombinase procedure to remove the resistant marker from primary transformant 792

was performed as previously described (Trouiller et al., 2006) with minor modifications. 793

Transformed tissue was grown as wildtype, and protoplast production and transformation was 794

performed using 20 µg of the plasmid pAct-Cre (Trouiller et al., 2006). Diluted protoplasts 795

suspensions were plated on cellophane coated medium (ca. 25,000 counted protoplasts per 9 cm 796

Petri dish) to avoid mixing of regenerated plants. Protoplast regeneration and test procedure were 797

performed as follows: 1) four days protoplast regeneration on PRMB medium; 2) four days 798

protoplast growth on BCDA medium; 3) individual plants transferred to fresh BCDA medium 799

and growth for eight days; and 4) replica plating of each individual plant unto BCDA- and 800

BCDA-medium supplemented with the appropriate antibiotic. Strains showing loss of antibiotic 801

resistance were selected and grown until sufficient tissue was available for PCR genotyping. 802

803

PCR genotyping of transformants 804

Each mutant was subjected to three rounds of PCR genotyping, except otherwise stated, 805

using the Phire Plant Direct PCR kit (Thermo Fisher Scientific) according to the manufacturer’s 806

instructions. The primer sequences used for genotyping each set of transformants are provided in 807

Supplemental Table S3. In the first round of genotyping, deletion mutants were genotyped for the 808

loss of genomic sequence, while in the case of knock-in mutants, we genotyped for the gain of 809



29

sequence. In the second PCR, we genotyped for proper 5’ and 3’ targeting at the locus using a 810

primer annealing within the DNA fragment used for transformation and a primer annealing 5’ or 811

3’ to the 5’ and 3’ TGSs, respectively. Finally, in the third PCR we genotyped for single-copy 812

insertions at the locus using primers annealing 5’ and 3’ to the 5’ and 3’ TGSs, respectively. 813

For dek1ΔlinkerG1 (Supplemental Fig. S1A), first genotyping was performed using primers 814

Δlinker_1F and Δlinker_1R to assess absence/presence of the Linker in HygR lines. Lines 815

without a detectable PCR product, presumed to have lost the Linker, were tested in the second 816

round of PCR genotyping to check for on-locus targeting using primers Δlinker_2F and 817

35S_rev1 for 5’ targeting, and Term_F and Δlinker_2R for 3’ targeting. Targeted single copy 818

insertion was assessed in the third PCR genotyping using primers Δlinker_2F and Δlinker_2R. 819

The same rationale was used for genotyping dek1Δlinker3G transformants (Supplemental Fig. 820

S2A) using the following primer sets; first genotyping: Δlinker_1F and Δlinker_1R; second 821

genotyping: SP_LOOP_inverse and 35S_rev1 for 5’ targeting, and Term_F and gDek1_rev for 3’ 822

targeting; third genotyping: SP_LOOP_inverse and gDek1_rev. In the case of dek1Δc-linker 823

(Supplemental Fig. S6A), first genotyping was performed using primers ARMseq7 and 824

ASP_PpCALP, showing a distinction between WT and transformed plants by a 478 bp reduction 825

in the PCR fragment size with respect to WT, corresponding to the size of the deleted fragment. 826

For second genotyping, primers TM2seq1 and 35S_rev1 for 5’ targeting, and Term_F and 827

ASP_PpCALP for 3’ targeting, were used, while TM2seq1 and ASP_PpCALP were used for third 828

genotyping, assessing single copy on-locus insertions. For dek1Δlg3 where screening of 829

transformed plants were based on phenotype, two rounds of PCR genotyping was performed 830

(Supplemental Fig. S4A); first PCR using primers ARMseq6 and ASP_PpARM_inf1 that anneal 831

within the targeting DNA, and secondly using primers TM2seq1 and ASP_PpCALP that anneal 832

outside the targeting DNA. Finally, for dek10 (Supplemental Fig. S7A), targeting was assessed 833

using primers Null_1F and Null_1R with PCR extension condition set to amplify only 834

non-transformed plants (due to the presence of the resistance cassette in the transformed lines). 835

G418R plants without a detectable WT band were assessed for on-locus targeting using primers 836

Null_2F and 35S-rev1 for 5’ targeting, and Term-F and Null_2R for 3’ targeting. Next, single 837

copy integration at the locus was assessed using primers Null_2F and Null_2R. 838

839



30

Molecular characterization of transformants 840

Genomic DNA for Southern Blot analysis was extracted using the Nucleon™ 841

PhytoPure™ Genomic DNA Extraction Kit (GE Healthcare). Southern Blot was 842

performed as described by Perroud and Quatrano (2006) using 1 µg DNA 843

per digestion. Probes were DIG-labelled using the DIG Probe PCR synthesis kit (Roche, 844

Indianapolis, USA) according to the manufacturer’s instructions. Template for PCR 845

amplification of probes were the corresponding DNA constructs used for transformation. The 846

sequence of the primers used to generate the probes are provide in Supplemental Table S3, and 847

the hybridization sites of each probe are schematically illustrated in Supplemental Figs. 848

S1A-S7A. 849

RT-PCR and DNA sequencing was used to analyze the mutant Pp DEK1 cDNA to verify 850

proper deletion/insertion and splicing of the transcript. Total RNA was isolated from protonema 851

tissue using the Plant RNeasy Kit (Qiagen). 500 ng DNaseI-treated total RNA was 852

reverse-transcribed using 100 U Superscript™ III Reverse Transcriptase (Invitrogen) primed 853

with 200 ng random hexamers at 55 °C for 60 min. Phusion® High-Fidelity DNA polymerase 854

was used to amplify the DEK1 cDNA target using primers Ex7-F and Ex30-R, annealing in exon 855

7 and 30, respectively. After Exo SAP treatment to remove unused primers, the PCR product was 856

sequenced with appropriate primers using BigDye® v3.1 chemistry (ABI) according to the SteP 857

method (Platt et al., 2007). Extension products were precipitated using sodium-acetate/ethanol, 858

dissolved in Hi-Di™ Formamide (ABI) and finally sequenced by Capillary Electrophoresis using 859

the 3130xL Genetic Analyzer. DNA sequences were analyzed using Genomic Workbench v7. 860

861

Real-Time Quantitative Reverse Transcription PCR (q-PCR) 862

q-PCR was performed to determine the transcript level of DEK1 in the P. patens mutants 863

relative to the WT control. RNA was extracted in two independent biological replicates using the 864

Plant RNeasy Kit (Qiagen) and cDNA was prepared as described previously. Transcript level 865

analysis was performed in duplicates using 0.2 µM forward and reverse primers and 1:4 diluted 866

cDNA with the HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis Biodyne) on the 867

7500 Fast Real-Time PCR Systems (ABI). qPCR cycling was performed as follows: 95 °C for 15 868

mins followed by 40 cycles of 95 °C for 30 secs, 58 °C for 20 sec and 72 °C for 32 sec for 869



31

amplification of all targets. The qPCR data were analyzed using the LinRegPCR software v11.0 870

(Ruijter et al., 2009) to obtain the threshold cycle (Ct) number and the mean efficiency values 871

(E) for each sample and target, respectively. Relative mRNA levels of the targets were 872

normalized to the transcript level of RIBOSOMAL PROTEIN S9 (RSB, Pp3c18_1350V3.1) (Xiao 873

et al., 2011). Fold expression in mutant samples were calculated relative to WT according to the 874

method of (Pfaffl, 2001). The following gene specific primer pairs were used for target 875

amplification: for DEK1, TMq-F and TMq-R; and for RSB, RSBq-F and RSBq-R. Each primer 876

pair generated a single, expected PCR product based on post-PCR dissociation analyses. 877

878

Accession numbers: 879

Supplemental Table S1 provides the accession numbers for all sequences used in this study. 880



32

Supplemental Material 881

882

Supplemental Data S1. Charophyte algae DEK1 Linker protein sequences used in this study. 883

Supplemental Data S2. Alignment of DEK1 Linker protein sequences in fasta format. 884

Supplemental Table S1. Accession numbers for the orthologous DEK1 Linker protein 885

sequences used in this study. 886

Supplemental Table S2. Amino acid sequence identity between each pair of DEK1 Linker 887

sequences. 888

Supplemental Table S3. List of oligonucleotide primers and their sequences used in this study. 889

Supplemental Figure S1. Vector construction, targeted DEK1 Linker deletion and molecular 890

characterization of the Physcomitrella patens dek1ΔlinkerG1/cre mutant line. 891

Supplemental Figure S2. Vector construction, targeted insertion and molecular characterization 892

of the Physcomitrella patens dek1ΔlinkerG3/cre#123 mutant line. 893

Supplemental Figure S3. Vector construction, targeted knock-in of the Physcomitrella patens 894

DEK1 Linker, PCR genotyping and gametophore morphology of complemented line. 895

Supplemental Fig. S4. Vector construction, targeted insertion of LinkerΔLG3 and molecular 896

characterization of the Physcomitrella patens dek1Δlg3 mutant line. 897

Supplemental Fig. S5. Vector construction, targeted knock-in of Physcomitrella patens, 898

Marchantia polymorpha and Arabidopsis thaliana DEK1 LG3, and PCR genotyping. 899

Supplemental Fig. S6. Vector construction, targeted insertion and molecular characterization of 900

the Physcomitrella patens dek1Δc-linker mutant line. 901

Supplemental Fig. S7. Vector construction, targeted insertion and molecular characterization of 902

the Physcomitrella patens dek10 mutant line. 903

904



33

ACKNOWLEDGMENTS 905

We thank warmly The Imaging Centre, NMBU for providing access to microscopes and 906

technical assistance with microscopic analysis, and Dr. Stefanie Reißmann and the AG Kahmann 907

at the MPI for terrestrial microbiology in Marburg for the use of their confocal microscopy 908

equipment. We also thank Dr. Rafi Ahmad at Hedmark University of Applied Sciences (HUAS) 909

for valuable help with modeling of the DEK1 LG3 domain. Finally, we thank Charles Delwiche 910

at University of Maryland, College Park, for sharing the charophyte DEK1 sequences with us. 911

The project was funded by grants from the Norwegian Research Council to O-A.O. and by funds 912

to W.J from HUAS. Support to A.E.A was from The Norwegian Research Council and HUAS. 913

914



34

FIGURE LEGENDS 915

Figure 1. Physomitrella patens DEK1 protein domain structure and DEK1 Linker sequence 916

conservation. A, Schematic representation of DEK1 domain structure. DEK1 MEM is composed 917

of two parts with multiple transmembrane (TM) segments disrupted by the Loop. The DEK1 918

Linker is divided into the three sub-segments N, LG3 and C. DEK1 calpain consists of CysPc, 919

the catalytic domain and C2L. Amino acid numbering is given above the DEK1 outline. B, Heat 920

map showing the degree of DEK1 Linker sequence conservation among 73 land plant species. 921

Percentage sequence identities are indicated in different colors, from blue (0 %; 922

insertions/deletions) to red (100%; absolute conserved positions). Amino acid numbering for the 923

different sub-segments of the P. patens DEK1 Linker is given above the upper figure in the 924

panel. The percentage above the heat map gives the sequence identity between the Arabidopsis 925

thaliana and P. patens DEK1 Linker sub-segments. C, Predicted structure of the P. patens DEK1 926

LG3 domain. I, Structural alignment of the predicted I-TASSER (blue), RaptorX (magnenta) and 927

Phyre2 (green) structures showing that the LG3 domain adopts a β-sandwich fold and that 928

individual servers predicted highly similar structures. II, Structural alignment between the 929

Phyre2 model and the 3FLP crystal structure (yellow) of native heptameric SAP-like pentraxin 930

from Limulus polyphemus. 931

Figure 2. DEK1 cDNA structure and expression analyses in wildtype, dek1Δlinker, dek1Δlg3, 932

dek1Δc-linker and dek10 lines. A, Schematic representation of the DEK1 coding sequence 933

architecture in wildtype and dek1 mutant plants. The truncated DEK1 variants are missing either 934

the Linker or the Linker sub-segments LG3 and C, while DEK10 harbors two point mutations 935

leading to a single amino acid replacement from Cys to Ser in the CysPc coding sequence of the 936

active site. Numberings identify deleted nucleotides (nt) in the individual truncated DEK1 937

variants. The codon TGC starting at the WT sequence in nt position 5341 (black star) is mutated 938

to TCT (white star) in DEK10. The position of the PCR primers (Ex7-F and Ex30-R) used in B 939

are indicated by arrows, and expected RT-PCR product sizes using this primer set are given. For 940

comparison, WT DEK1 is also shown. B, RT-PCR analysis of DEK1 transcripts in WT and dek1 941

mutant plants using primers Ex7-F and Ex30-R showing PCR amplicons of the expected sizes. 942

M=marker, NC=negative control. C, Real-time Quantitative RT-PCR analysis of relative DEK1 943

transcript levels in dek1 mutant plants at two time points (6 and 14 days). The graph shows the 944



35

mean values of fold change in the dek1 mutants relative to WT, which was set to 1, and 945

normalized to the average transcript level of RIBOSOMAL PROTEIN S9 (RSB; 946

Pp3c18_1350V3.1) used as internal reference. Bars indicate ±SE of two biological replicates. 947

Figure 3. Wildtype and dek1 mutant phenotypes. A, WT plants, B-G, dek1 mutants. The 948

phenotypes at different developmental stages are showed in columns 1 to 8. Column 1 shows 949

number of buds per 15-cells filament (n=100). Column 2 buds at 4 cell stage. Arrowheads 950

indicate the first oblique division of the bud initial. Arrows indicate the plane of the first division 951

in the bud apical cell. Blue dots mark the bud apical initial. Yellow dots mark apical lateral cells. 952

Brown dots mark basal lateral cells. Column 3 shows bud development at the six or more cell 953

stage. Arrowheads indicate position of the initial oblique division. Blue dots mark bud apical 954

cells. Yellow dots mark apical lateral cells (forming a hair in A and E). Green dots in E marks a 955

phyllid initial cell. Brown dots mark basal lateral cells. Column 4 details young gametophores 956

with primordial phyllids in A and E. Green dots mark phyllid initials. Yellow dots mark a hair. 957

Please note that the latter structures are not formed in B and G. Column 5 shows leafy 958

gametophores in A and E and reduced stems lacking phyllids in C and F. These structures are not 959

formed in B, D and G. Column 6 displays morphology of a phyllid isolated from the middle part 960

of a gametophores. Column 7 represents morphology of archegonia at later stages of 961

development. The archegonium apex is opened and a neck canal is marked by brown 962

pigmentation in A. The neck remains closed and is distorted in E. Column 8 shows a mature 963

sporophyte. Bar: 50 μm (column 2 and 3; A7, E7); 100 μm (column 4); 1 mm (A5, E5); 500 μm 964

(C5, F5, A8); 200 μm (A6, E6). 965

Figure 4. Number of buds initiated in Physcomitrella patens wild type and dek1 mutant plants. 966

The graph shows the average number of buds per 15-cell filament (n=100) in wild type, Δdek1, 967

dek1Δlinker, dek1Δlg3, dek1Δc-linker and dek10 lines. Lines over bars indicate no significant 968

differences, paired t-test. WT = wild type. Bars indicate ±SE. 969

Figure 5. Phyllid and reproductive organ morphology in WT and dek1lg3. A, WT phyllids 970

isolated from the basal, middle and apical part of the gametophore, respectively. Position of the 971

isolated phyllid on gametophore is marked by arrowheads in the right lower insertions. B, 972

dek1lg3 phyllids isolated from the basal, middle and apical part of the gametophore, respectively. 973

Position of the isolated phyllid on gametophore is marked by arrowheads in the right lower 974



36

insertions. C, Young WT archegonium with highlighted cell walls in the neck. D, Examples of 975

WT archegonia at the later stages of development. Note open apices and brown pigmentation 976

marking the neck canal. E, WT gametophore with sporophyte. Antheridium is shown in the 977

lower left insertion. F, Young dek1lg3 archegonium with highlighted cell walls in the neck. G, 978

dek1lg3 archegonia at the later stages of development. Note closed and deformed necks and 979

patchy distribution of the brown pigmentation that reflects defects in the neck canal 980

differentiation. H, dek1lg3 gametophores cultivated under the sporogenesis conditions. 981

Sporophytes are not formed. Antheridium is shown in the lower left insertion. Bars: 500 μm (A 982

and B); 50 μm (C, D, F, G); 1 mm (E, H). 983

Figure 6. DEK1 LG3 complementation. A, WT gametophore. B, dek1lg3 gametophore. C, 984

Gametophore of the dek1lg3 strain after re-targeting of the P. patens DEK1-LG3 sequence. D, 985

dek1Mplg3 gametophore - dek1lg3 after introduction of the M. polymorpha DEK1 LG3 986

sequence. Bar: 1mm. 987

988

989



37

REFERENCES 990

Aoyama T, Hiwatashi Y, Shigyo M, Kofuji R, Kubo M, Ito M, Hasebe M (2012) AP2-type 991

transcription factors determine stem cell identity in the moss Physcomitrella patens. 992

Development 139: 3120-3129 993

Arthur JSC, Gauthier S, Elce JS (1995) ACTIVE-SITE RESIDUES IN M-CALPAIN - 994 IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS. Febs Letters 368: 397-400 995

996 Becraft PW, Li KJ, Dey N, Asuncion-Crabb Y (2002) The maize dek1 gene functions in 997

embryonic pattern formation and cell fate specification. Development 129: 5217-5225 998

Chandra NR, Prabu MM, Suguna K, Vijayan M (2001) Structural similarity and functional 999

diversity in proteins containing the legume lectin fold. Protein Engineering 14: 857-866 1000

Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) The moss 1001

Physcomitrella patens: a novel model system for plant development and genomic studies. 1002

Cold Spring Harbor protocols 2009: pdb.emo115-pdb.emo115 1003

Demko V, Perroud P-F, Johansen W, Delwiche CF, Cooper ED, Remme P, Ako AE, Kugler KG, 1004

Mayer KFX, Quatrano R, Olsen O-A (2014) Genetic Analysis of DEFECTIVE 1005

KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella 1006

patens. Plant Physiology 166: 903-U684 1007

Du Clos TW (2013) Pentraxins: structure, function, and role in inflammation. ISRN 1008

inflammation 2013: 379040-379040 1009

Festucci-Buselli RA, Otoni WC, Joshi CP (2007) Structure, organization, and functions of 1010

cellulose synthase complexes in higher plants. Brazilian Journal of Plant Physiology 19: 1011

1-13 1012

Frank MH, Edwards MB, Schultz ER, McKain MR, Fei ZJ, Sorensen I, Rose JKC, Scanlon MJ 1013

(2015) Dissecting the molecular signatures of apical cell-type shoot meristems from two 1014

ancient land plant lineages. New Phytologist 207: 893-904 1015

Goss CA, Brockmann DJ, Bushoven JT, Roberts AW (2012) A CELLULOSE SYNTHASE 1016

(CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella 1017

patens. Planta 235: 1355-1367 1018

Harrison CJ (2015) Shooting through time: new insights from transcriptomic data. Trends in 1019

Plant Science 20: 468-470 1020

Jacob-Wilk D, Kurek I, Hogan P, Delmer DP (2006) The cotton fiber zinc-binding domain of 1021



38

cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in 1022

vivo. Proceedings of the National Academy of Sciences of the United States of America 1023

103: 12191-12196 1024

Johnson KL, Faulkner C, Jeffree CE, Ingram GC (2008) The Phytocalpain Defective Kernel 1 Is 1025

a Novel Arabidopsis Growth Regulator Whose Activity Is Regulated by Proteolytic 1026

Processing. Plant Cell 20: 2619-2630 1027

Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: 1028

Improvements in Performance and Usability. Molecular Biology and Evolution 30: 1029

772-780 1030

Khorchid A, Ikura M (2002) How calpain is activated by calcium. Nature Structural Biology 9: 1031

239-241 1032

Kofuji R, Hasebe M (2014) Eight types of stem cells in the life cycle of the moss Physcomitrella 1033

patens. Current Opinion in Plant Biology 17: 13-21 1034

Landberg K, Pederson ERA, Viaene T, Bozorg B, Friml J, Jonsson H, Thelander M, Sundberg E 1035

(2013) The Moss Physcomitrella patens Reproductive Organ Development Is Highly 1036

Organized, Affected by the Two SHI/STY Genes and by the Level of Active Auxin in the 1037

SHI/STY Expression Domain. Plant Physiology 162: 1406-1419 1038

Liang Z, Demko V, Wilson RC, Johnson KA, Ahmad R, Perroud P-F, Quatrano R, Zhao S, 1039

Shalchian-Tabrizi K, Otegui MS, Olsen O-A, Johansen W (2013) The catalytic domain 1040

CysPc of the DEK1 calpain is functionally conserved in land plants. Plant Journal 75: 1041

742-754 1042

Lid SE, Gruis D, Jung R, Lorentzen JA, Ananiev E, Chamberlin M, Niu XM, Meeley R, Nichols 1043

S, Olsen OA (2002) The defective kernel 1 (dek1) gene required for aleurone cell 1044

development in the endosperm of maize grains encodes a membrane protein of the 1045

calpain gene superfamily. Proceedings of the National Academy of Sciences of the 1046

United States of America 99: 5460-5465 1047

Lid SE, Olsen L, Nestestog R, Aukerman M, Brown RC, Lemmon B, Mucha M, 1048

Opsahl-Sorteberg HG, Olsen OA (2005) Mutation in the Arabidopisis thaliana DEK1 1049

calpain gene perturbs endosperm and embryo development while over-expression affects 1050

organ development globally. Planta 221: 339-351 1051

Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia ZC, Davies PL (2002) A Ca2+ switch aligns 1052



39

the active site of calpain. Cell 108: 649-660 1053

Olsen O-A, Perroud P-F, Johansen W, Demko V (2015) DEK1; missing piece in puzzle of plant 1054

development. Trends in Plant Science 20: 70-71 1055

Perroud P-F, Cove DJ, Quatrano RS, McDaniel SF (2011) An experimental method to facilitate 1056

the identification of hybrid sporophytes in the moss Physcomitrella patens using 1057

fluorescent tagged lines. New Phytologist 191: 301-306 1058

Perroud P-F, Demko V, Johansen W, Wilson RC, Olsen O-A, Quatrano RS (2014) Defective 1059

Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens. New 1060

Phytologist 203: 794-804 1061

Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. 1062

Nucleic Acids Research 29 1063

Pires ND, Dolan L (2012) Morphological evolution in land plants: new designs with old genes. 1064

Philosophical Transactions of the Royal Society B-Biological Sciences 367: 508-518 1065

Prigge MJ, Bezanilla M (2010) Evolutionary crossroads in developmental biology: 1066

Physcomitrella patens. Development 137: 3535-3543 1067

Rudenko G, Hohenester E, Muller YA (2001) LG/LNS domains: multiple functions - one 1068

business end? Trends in Biochemical Sciences 26: 363-368 1069

Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman 1070

AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of 1071

quantitative PCR data. Nucleic Acids Research 37 1072

Saunders MJ, Hepler PK (1982) Calcium ionophore a23187 stimulates cytokinin-like mitosis in 1073

funaria. Science 217: 943-945 1074

Schaefer DG, Delacote F, Charlot F, Vrielynck N, Guyon-Debast A, Le Guin S, Neuhaus JM, 1075

Doutriaux MP, Nogue F (2010) RAD51 loss of function abolishes gene targeting and 1076

de-represses illegitimate integration in the moss Physcomitrella patens. DNA Repair 9: 1077

526-533 1078

Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant 1079

Journal 11: 1195-1206 1080

Shinkai-Ouchi F, Koyama S, Ono Y, Hata S, Ojima K, Shindo M, duVerle D, Ueno M, Kitamura 1081

F, Doi N, Takigawa I, Mamitsuka H, Sorimachi H (2016) Predictions of Cleavability of 1082

Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using 1083



40

Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array. 1084

Molecular & Cellular Proteomics 15: 1262-1280 1085

Sorimachi H, Mamitsuka H, Ono Y (2012) Understanding the substrate specificity of 1086

conventional calpains. Biological Chemistry 393: 853-871 1087

Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, 1088

Sorimachi H, Bourenkow G, Bartunik H, Suzuki K, Bode W (2000) The crystal structure 1089

of calcium-free human m-calpain suggests an electrostatic switch mechanism for 1090

activation by calcium. Proceedings of the National Academy of Sciences of the United 1091

States of America 97: 588-592 1092

Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, Friedrich P 1093

(2004) On the sequential determinants of calpain cleavage. Journal of Biological 1094

Chemistry 279: 20775-20785 1095

Trouiller B, Schaefer DG, Charlot F, Nogue F (2006) MSH2 is essential for the preservation of 1096

genome integrity and prevents homeologous recombination in the moss Physcomitrella 1097

patens. Nucleic Acids Research 34: 232-242 1098

Wang CX, Barry JK, Min Z, Tordsen G, Rao AG, Olsen OA (2003) The calpain domain of the 1099

maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine 1100

proteinase. Journal of Biological Chemistry 278: 34467-34474 1101

Wendt A, Thompson VF, Goll DE (2004) Interaction of calpastatin with calpain: a review. 1102

Biological Chemistry 385: 465-472 1103

Xiao L, Wang H, Wan P, Kuang T, He Y (2011) Genome-wide transcriptome analysis of 1104

gametophyte development in Physcomitrella patens. Bmc Plant Biology 11 1105

Yan N (2013) Structural advances for the major facilitator superfamily (MFS) transporters. 1106

Trends in Biochemical Sciences 38: 151-159 1107

Zhao S, Liang Z, Demko V, Wilson R, Johansen W, Olsen O-A, Shalchian-Tabrizi K (2012) 1108

Massive expansion of the calpain gene family in unicellular eukaryotes. Bmc 1109

Evolutionary Biology 12 1110

1111



Figure 1. Physomitrella patens DEK1 protein domain structure and DEK1 Linker sequence conservation. A, Schematic

representation of DEK1 domain structure. DEK1 MEM is composed of two parts with multiple transmembrane (TM)

segments disrupted by the Loop. The DEK1 Linker is divided into the three sub-segments N, LG3 and C. DEK1 calpain

consists of CysPc, the catalytic domain and C2L. Amino acid numbering is given above the DEK1 outline. B, Heat map

showing the degree of DEK1 Linker sequence conservation among 73 land plant species. Percentage sequence identities are

indicated in different colors, from blue (0 %; insertions/deletions) to red (100%; absolute conserved positions). Amino acid

numbering for the different sub-segments of the P. patens DEK1 Linker is given above the upper figure in the panel. The

percentage above the heat map gives the sequence identity between the Arabidopsis thaliana and P. patens DEK1 Linker

sub-segments. C, Predicted structure of the P. patens DEK1 LG3 domain. I, Structural alignment of the predicted I-TASSER

(blue), RaptorX (magnenta) and Phyre2 (green) structures showing that the LG3 domain adopts a b-sandwich fold and that

individual servers predicted highly similar structures. II, Structural alignment between the Phyre2 model and the 3FLP

crystal structure (yellow) of native heptameric SAP-like pentraxin from Limulus polyphemus.



WT

Fold

ch

ange

Figure 2

B C

Figure 2. DEK1 cDNA structure and expression analyses in wildtype, dek1Dlinker, dek1Dlg3, dek1Dc-linker and

dek10 lines. A, Schematic representation of the DEK1 coding sequence architecture in wildtype and dek1 mutant

plants. The truncated DEK1 variants are missing either the Linker or the Linker sub-segments LG3 and C, while

DEK10 harbors two point mutations leading to a single amino acid replacement from Cys to Ser in the CysPc

coding sequence of the active site. Numberings identify deleted nucleotides (nt) in the individual truncated DEK1

variants. The codon TGC starting at the WT sequence in nt position 5341 (black star) is mutated to TCT (white

star) in DEK10. The position of the PCR primers (Ex7-F and Ex30-R) used in B are indicated by arrows, and

expected RT-PCR product sizes using this primer set are given. For comparison, WT DEK1 is also shown. B, RT-

PCR analysis of DEK1 transcripts in WT and dek1 mutant plants using primers Ex7-F and Ex30-R showing PCR

amplicons of the expected sizes. M=marker, NC=negative control. C, Real-time Quantitative RT-PCR analysis of

relative DEK1 transcript levels in dek1 mutant plants at two time points (6 and 14 days). The graph shows the mean

values of fold change in the dek1 mutants relative to WT, which was set to 1, and normalized to the average

transcript level of RIBOSOMAL PROTEIN S9 (RSB; Pp3c18_1350V3.1) used as internal reference. Bars indicate

±SE of two biological replicates.



A

B

C

D

E

F

G

WT

Δdek1

dek1Δloop

dek1Δlinker

dek1Δlg3

dek1Δc-linker

dek10

Perroud et al.,

2014

Demko et al.,

2014

1.1±0.3

3.5±1.2

1.9±0.2

2.0±0.6

3.3±1.2

1.9±0.8

1.2±0.4

1 2 3 4 5 6 7 8

Figure 3. Wildtype and dek1 mutant phenotypes. A, WT plants, B-G, dek1 mutants. The phenotypes at different

developmental stages are showed in columns 1 to 8. Column 1 shows number of buds per 15-cells filament (n=100).

Column 2 buds at 4 cell stage. Arrowheads indicate the first oblique division of the bud initial. Arrows indicate the

plane of the first division in the bud apical cell. Blue dots mark the bud apical initial. Yellow dots mark apical lateral

cells. Brown dots mark basal lateral cells. Column 3 shows bud development at the six or more cell stage. Arrowheads

indicate position of the initial oblique division. Blue dots mark bud apical cells. Yellow dots mark apical lateral cells

(forming a hair in A and E). Green dots in E marks a phyllid initial cell. Brown dots mark basal lateral cells. Column 4

details young gametophores with primordial phyllids in A and E. Green dots mark phyllid initials. Yellow dots mark a

hair. Please note that the latter structures are not formed in B and G. Column 5 shows leafy gametophores in A and E

and reduced stems lacking phyllids in C and F. These structures are not formed in B, D and G. Column 6 displays

morphology of a phyllid isolated from the middle part of a gametophores. Column 7 represents morphology of

archegonia at later stages of development. The archegonium apex is opened and a neck canal is marked by brown

pigmentation in A. The neck remains closed and is distorted in E. Column 8 shows a mature sporophyte. Bar: 50 μm

(column 2 and 3; A7, E7); 100 μm (column 4); 1 mm (A5, E5); 500 μm (C5, F5, A8); 200 μm (A6, E6). www.plantphysiol.orgon February 13, 2018 - Published by Downloaded from

Copyright © 2016 American Society of Plant Biologists. All rights reserved.


WT

1

2

3

4

5

p = 0.10

p = 0.17

p = 0.20

Num

ber

of

bud

s p

er 1

5-c

ell

fila

men

t

Figure 4

Figure 4. Number of buds initiated in Physcomitrella patens wild type

and dek1 mutant plants. The graph shows the average number of buds

per 15-cell filament (n=100) in wild type, Ddek1, dek1Dlinker,

dek1Dlg3, dek1Dc-linker and dek10 lines. Lines over bars indicate no

significant differences, paired t-test. WT = wild type. Bars indicate

±SE.



Figure 5. Phyllid and reproductive organ morphology in WT and dek1Δlg3. A, WT phyllids isolated from the basal, middle and apical part of the gametophore, respectively. Position of the isolated phyllid on gametophore is marked by arrowheads in the right lower insertions. B, dek1Δlg3 phyllids isolated from the basal, middle and apical part of the gametophore, respectively. Position of the isolated phyllid on gametophore is marked by arrowheads in the right lower insertions. C, Young WT archegonium with highlighted cell walls in the neck. D, Examples of WT archegonia at the later stages of development. Note open apices and brown pigmentation marking the neck canal. E, WT gametophore with sporophyte. Antheridium is shown in the lower left insertion. F, Young dek1Δlg3 archegonium with highlighted cell walls in the neck. G, dek1Δlg3 archegonia at the later stages of development. Note closed and deformed necks and patchy distribution of the brown pigmentation that reflects defects in the neck canal differentiation. H, dek1Δlg3 gametophores cultivated under the sporogenesis conditions. Sporophytes are not formed. Antheridium is shown in the lower left insertion. Bars: 500 µm (A and B); 50 µm (C, D, F, G); 1 mm (E, H).



WT dek1Δlg3 PpLG3 MpLG3 A B C D

Figure 6. DEK1 LG3 complementation. A, WT gametophore. B, dek1Δlg3 gametophore. C, Gametophore of the dek1Δlg3 strain after re-targeting of the P. patens DEK1-LG3 sequence. D, dek1Mplg3 gametophore - dek1Δlg3 after introduction of the M. polymorpha DEK1 LG3 sequence. Bar: 1mm.



Parsed CitationsAoyama T, Hiwatashi Y, Shigyo M, Kofuji R, Kubo M, Ito M, Hasebe M (2012) AP2-type transcription factors determine stem cellidentity in the moss Physcomitrella patens. Development 139: 3120-3129

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Arthur JSC, Gauthier S, Elce JS (1995) ACTIVE-SITE RESIDUES IN M-CALPAIN - IDENTIFICATION BY SITE-DIRECTEDMUTAGENESIS. Febs Letters 368: 397-400


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


Chandra NR, Prabu MM, Suguna K, Vijayan M (2001) Structural similarity and functional diversity in proteins containing the legumelectin fold. Protein Engineering 14: 857-866


Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) The moss Physcomitrella patens: a novelmodel system for plant development and genomic studies. Cold Spring Harbor protocols 2009: pdb.emo115-pdb.emo115


Demko V, Perroud P-F, Johansen W, Delwiche CF, Cooper ED, Remme P, Ako AE, Kugler KG, Mayer KFX, Quatrano R, Olsen O-A(2014) Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella patens.Plant Physiology 166: 903-U684


Du Clos TW (2013) Pentraxins: structure, function, and role in inflammation. ISRN inflammation 2013: 379040-379040Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Festucci-Buselli RA, Otoni WC, Joshi CP (2007) Structure, organization, and functions of cellulose synthase complexes in higherplants. Brazilian Journal of Plant Physiology 19: 1-13


Frank MH, Edwards MB, Schultz ER, McKain MR, Fei ZJ, Sorensen I, Rose JKC, Scanlon MJ (2015) Dissecting the molecularsignatures of apical cell-type shoot meristems from two ancient land plant lineages. New Phytologist 207: 893-904


Goss CA, Brockmann DJ, Bushoven JT, Roberts AW (2012) A CELLULOSE SYNTHASE (CESA) gene essential for gametophoremorphogenesis in the moss Physcomitrella patens. Planta 235: 1355-1367


Harrison CJ (2015) Shooting through time: new insights from transcriptomic data. Trends in Plant Science 20: 468-470Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jacob-Wilk D, Kurek I, Hogan P, Delmer DP (2006) The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypiumhirsutum displays rapid turnover in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States ofAmerica 103: 12191-12196


Johnson KL, Faulkner C, Jeffree CE, Ingram GC (2008) The Phytocalpain Defective Kernel 1 Is a Novel Arabidopsis GrowthRegulator Whose Activity Is Regulated by Proteolytic Processing. Plant Cell 20: 2619-2630



http://www.ncbi.nlm.nih.gov/pubmed?term=Aoyama T%2C Hiwatashi Y%2C Shigyo M%2C Kofuji R%2C Kubo M%2C Ito M%2C Hasebe M %282012%29 AP2%2Dtype transcription factors determine stem cell identity in the moss Physcomitrella patens%2E Development 139%3A 3120%2D3129&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Aoyama T, Hiwatashi Y, Shigyo M, Kofuji R, Kubo M, Ito M, Hasebe M (2012) AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens. Development 139: 3120-3129

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Aoyama&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Aoyama&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Arthur JSC%2C Gauthier S%2C Elce JS %281995%29 ACTIVE%2DSITE RESIDUES IN M%2DCALPAIN %2D IDENTIFICATION BY SITE%2DDIRECTED MUTAGENESIS%2E Febs Letters 368%3A 397%2D400&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Arthur JSC, Gauthier S, Elce JS (1995) ACTIVE-SITE RESIDUES IN M-CALPAIN - IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS. Febs Letters 368: 397-400

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Arthur&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=ACTIVE-SITE RESIDUES IN M-CALPAIN - IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=ACTIVE-SITE RESIDUES IN M-CALPAIN - IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Arthur&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Becraft PW%2C Li KJ%2C Dey N%2C Asuncion%2DCrabb Y %282002%29 The maize dek1 gene functions in embryonic pattern formation and cell fate specification%2E Development 129%3A 5217%2D5225&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Becraft PW, Li KJ, Dey N, Asuncion-Crabb Y (2002) The maize dek1 gene functions in embryonic pattern formation and cell fate specification. Development 129: 5217-5225

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Becraft&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The maize dek1 gene functions in embryonic pattern formation and cell fate specification&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The maize dek1 gene functions in embryonic pattern formation and cell fate specification&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Becraft&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chandra NR%2C Prabu MM%2C Suguna K%2C Vijayan M %282001%29 Structural similarity and functional diversity in proteins containing the legume lectin fold%2E Protein Engineering 14%3A 857%2D866&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chandra NR, Prabu MM, Suguna K, Vijayan M (2001) Structural similarity and functional diversity in proteins containing the legume lectin fold. Protein Engineering 14: 857-866

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chandra&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Structural similarity and functional diversity in proteins containing the legume lectin fold&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Structural similarity and functional diversity in proteins containing the legume lectin fold&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chandra&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cove DJ%2C Perroud P%2DF%2C Charron AJ%2C McDaniel SF%2C Khandelwal A%2C Quatrano RS %282009%29 The moss Physcomitrella patens%3A a novel model system for plant development and genomic studies%2E Cold Spring Harbor protocols 2009%3A pdb%2Eemo115%2Dpdb%2Eemo115&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harbor protocols 2009: pdb.emo115-pdb.emo115

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cove&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The moss Physcomitrella patens: a novel model system for plant development and genomic studies&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The moss Physcomitrella patens: a novel model system for plant development and genomic studies&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cove&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Demko V%2C Perroud P%2DF%2C Johansen W%2C Delwiche CF%2C Cooper ED%2C Remme P%2C Ako AE%2C Kugler KG%2C Mayer KFX%2C Quatrano R%2C Olsen O%2DA %282014%29 Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three%2DDimensional Body Patterning in Physcomitrella patens%2E Plant Physiology 166%3A 903%2DU684&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Demko V, Perroud P-F, Johansen W, Delwiche CF, Cooper ED, Remme P, Ako AE, Kugler KG, Mayer KFX, Quatrano R, Olsen O-A (2014) Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella patens. Plant Physiology 166: 903-U684

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Demko&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Genetic Analysis of DEFECTIVE KERNEL1 Loop Function in Three-Dimensional Body Patterning in Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Demko&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Du Clos TW %282013%29 Pentraxins%3A structure%2C function%2C and role in inflammation%2E ISRN inflammation 2013%3A 379040%2D379040&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Du Clos TW (2013) Pentraxins: structure, function, and role in inflammation. ISRN inflammation 2013: 379040-379040

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Du&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Pentraxins: structure, function, and role in inflammation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Pentraxins: structure, function, and role in inflammation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Du&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Festucci%2DBuselli RA%2C Otoni WC%2C Joshi CP %282007%29 Structure%2C organization%2C and functions of cellulose synthase complexes in higher plants%2E Brazilian Journal of Plant Physiology 19%3A 1%2D13&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Festucci-Buselli RA, Otoni WC, Joshi CP (2007) Structure, organization, and functions of cellulose synthase complexes in higher plants. Brazilian Journal of Plant Physiology 19: 1-13

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Festucci-Buselli&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Structure, organization, and functions of cellulose synthase complexes in higher plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Structure, organization, and functions of cellulose synthase complexes in higher plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Festucci-Buselli&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Frank MH%2C Edwards MB%2C Schultz ER%2C McKain MR%2C Fei ZJ%2C Sorensen I%2C Rose JKC%2C Scanlon MJ %282015%29 Dissecting the molecular signatures of apical cell%2Dtype shoot meristems from two ancient land plant lineages%2E New Phytologist 207%3A 893%2D904&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Frank MH, Edwards MB, Schultz ER, McKain MR, Fei ZJ, Sorensen I, Rose JKC, Scanlon MJ (2015) Dissecting the molecular signatures of apical cell-type shoot meristems from two ancient land plant lineages. New Phytologist 207: 893-904

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Frank&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Dissecting the molecular signatures of apical cell-type shoot meristems from two ancient land plant lineages&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Dissecting the molecular signatures of apical cell-type shoot meristems from two ancient land plant lineages&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Frank&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Goss CA%2C Brockmann DJ%2C Bushoven JT%2C Roberts AW %282012%29 A CELLULOSE SYNTHASE %28CESA%29 gene essential for gametophore morphogenesis in the moss Physcomitrella patens%2E Planta 235%3A 1355%2D1367&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Goss CA, Brockmann DJ, Bushoven JT, Roberts AW (2012) A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens. Planta 235: 1355-1367

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Goss&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Goss&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Harrison CJ %282015%29 Shooting through time%3A new insights from transcriptomic data%2E Trends in Plant Science 20%3A 468%2D470&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Harrison CJ (2015) Shooting through time: new insights from transcriptomic data. Trends in Plant Science 20: 468-470

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Harrison&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Shooting through time: new insights from transcriptomic data&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Shooting through time: new insights from transcriptomic data&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Harrison&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Jacob%2DWilk D%2C Kurek I%2C Hogan P%2C Delmer DP %282006%29 The cotton fiber zinc%2Dbinding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo%2E Proceedings of the National Academy of Sciences of the United States of America 103%3A 12191%2D12196&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Jacob-Wilk D, Kurek I, Hogan P, Delmer DP (2006) The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America 103: 12191-12196

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jacob-Wilk&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jacob-Wilk&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Johnson KL%2C Faulkner C%2C Jeffree CE%2C Ingram GC %282008%29 The Phytocalpain Defective Kernel 1 Is a Novel Arabidopsis Growth Regulator Whose Activity Is Regulated by Proteolytic Processing%2E Plant Cell 20%3A 2619%2D2630&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=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

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Johnson&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The Phytocalpain Defective Kernel 1 Is a Novel Arabidopsis Growth Regulator Whose Activity Is Regulated by Proteolytic Processing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The Phytocalpain Defective Kernel 1 Is a Novel Arabidopsis Growth Regulator Whose Activity Is Regulated by Proteolytic Processing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Johnson&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1


Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance andUsability. Molecular Biology and Evolution 30: 772-780


Khorchid A, Ikura M (2002) How calpain is activated by calcium. Nature Structural Biology 9: 239-241Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kofuji R, Hasebe M (2014) Eight types of stem cells in the life cycle of the moss Physcomitrella patens. Current Opinion in PlantBiology 17: 13-21


Landberg K, Pederson ERA, Viaene T, Bozorg B, Friml J, Jonsson H, Thelander M, Sundberg E (2013) The Moss Physcomitrellapatens Reproductive Organ Development Is Highly Organized, Affected by the Two SHI/STY Genes and by the Level of ActiveAuxin in the SHI/STY Expression Domain. Plant Physiology 162: 1406-1419


Liang Z, Demko V, Wilson RC, Johnson KA, Ahmad R, Perroud P-F, Quatrano R, Zhao S, Shalchian-Tabrizi K, Otegui MS, Olsen O-A,Johansen W (2013) The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants. Plant Journal 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 defectivekernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein ofthe calpain gene superfamily. Proceedings of the National Academy of Sciences of the United States of America 99: 5460-5465


Lid SE, Olsen L, Nestestog R, Aukerman M, Brown RC, Lemmon B, Mucha M, Opsahl-Sorteberg HG, Olsen OA (2005) Mutation inthe Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organdevelopment globally. Planta 221: 339-351


Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia ZC, Davies PL (2002) A Ca2+ switch aligns the active site of calpain. Cell 108: 649-660


Olsen O-A, Perroud P-F, Johansen W, Demko V (2015) DEK1; missing piece in puzzle of plant development. Trends in PlantScience 20: 70-71


Perroud P-F, Cove DJ, Quatrano RS, McDaniel SF (2011) An experimental method to facilitate the identification of hybridsporophytes in the moss Physcomitrella patens using fluorescent tagged lines. New Phytologist 191: 301-306


Perroud P-F, Demko V, Johansen W, Wilson RC, Olsen O-A, Quatrano RS (2014) Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens. New Phytologist 203: 794-804


Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Pires ND, Dolan L (2012) Morphological evolution in land plants: new designs with old genes. Philosophical Transactions of theRoyal Society B-Biological Sciences 367: 508-518



http://www.ncbi.nlm.nih.gov/pubmed?term=Katoh K%2C Standley DM %282013%29 MAFFT Multiple Sequence Alignment Software Version 7%3A Improvements in Performance and Usability%2E Molecular Biology and Evolution 30%3A 772%2D780&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772-780

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Katoh&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Katoh&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Khorchid A%2C Ikura M %282002%29 How calpain is activated by calcium%2E Nature Structural Biology 9%3A 239%2D241&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Khorchid A, Ikura M (2002) How calpain is activated by calcium. Nature Structural Biology 9: 239-241

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Khorchid&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=How calpain is activated by calcium&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=How calpain is activated by calcium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Khorchid&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kofuji R%2C Hasebe M %282014%29 Eight types of stem cells in the life cycle of the moss Physcomitrella patens%2E Current Opinion in Plant Biology 17%3A 13%2D21&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kofuji R, Hasebe M (2014) Eight types of stem cells in the life cycle of the moss Physcomitrella patens. Current Opinion in Plant Biology 17: 13-21

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kofuji&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Eight types of stem cells in the life cycle of the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Eight types of stem cells in the life cycle of the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kofuji&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Landberg K%2C Pederson ERA%2C Viaene T%2C Bozorg B%2C Friml J%2C Jonsson H%2C Thelander M%2C Sundberg E %282013%29 The Moss Physcomitrella patens Reproductive Organ Development Is Highly Organized%2C Affected by the Two SHI%2FSTY Genes and by the Level of Active Auxin in the SHI%2FSTY Expression Domain%2E Plant Physiology 162%3A 1406%2D1419&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Landberg K, Pederson ERA, Viaene T, Bozorg B, Friml J, Jonsson H, Thelander M, Sundberg E (2013) The Moss Physcomitrella patens Reproductive Organ Development Is Highly Organized, Affected by the Two SHI/STY Genes and by the Level of Active Auxin in the SHI/STY Expression Domain. Plant Physiology 162: 1406-1419

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Landberg&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The Moss Physcomitrella patens Reproductive Organ Development Is Highly Organized, Affected by the Two SHI/STY Genes and by the Level of Active Auxin in the SHI/STY Expression Domain&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The Moss Physcomitrella patens Reproductive Organ Development Is Highly Organized, Affected by the Two SHI/STY Genes and by the Level of Active Auxin in the SHI/STY Expression Domain&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Landberg&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Liang Z%2C Demko V%2C Wilson RC%2C Johnson KA%2C Ahmad R%2C Perroud P%2DF%2C Quatrano R%2C Zhao S%2C Shalchian%2DTabrizi K%2C Otegui MS%2C Olsen O%2DA%2C Johansen W %282013%29 The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants%2E Plant Journal 75%3A 742%2D754&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Liang Z, Demko V, Wilson RC, Johnson KA, Ahmad R, Perroud P-F, Quatrano R, Zhao S, Shalchian-Tabrizi K, Otegui MS, Olsen O-A, Johansen W (2013) The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants. Plant Journal 75: 742-754

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liang&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lid SE%2C Gruis D%2C Jung R%2C Lorentzen JA%2C Ananiev E%2C Chamberlin M%2C Niu XM%2C Meeley R%2C Nichols S%2C Olsen OA %282002%29 The defective kernel 1 %28dek1%29 gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily%2E Proceedings of the National Academy of Sciences of the United States of America 99%3A 5460%2D5465&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=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. Proceedings of the National Academy of Sciences of the United States of America 99: 5460-5465

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lid&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=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&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=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&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lid&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lid SE%2C Olsen L%2C Nestestog R%2C Aukerman M%2C Brown RC%2C Lemmon B%2C Mucha M%2C Opsahl%2DSorteberg HG%2C Olsen OA %282005%29 Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over%2Dexpression affects organ development globally%2E Planta 221%3A 339%2D351&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=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: 339-351

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lid&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lid&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Moldoveanu T%2C Hosfield CM%2C Lim D%2C Elce JS%2C Jia ZC%2C Davies PL %282002%29 A Ca2%2B switch aligns the active site of calpain%2E Cell 108%3A 649%2D660&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia ZC, Davies PL (2002) A Ca2+ switch aligns the active site of calpain. Cell 108: 649-660

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Moldoveanu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A Ca2+ switch aligns the active site of calpain&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A Ca2+ switch aligns the active site of calpain&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Moldoveanu&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Olsen O%2DA%2C Perroud P%2DF%2C Johansen W%2C Demko V %282015%29 DEK1%3B missing piece in puzzle of plant development%2E Trends in Plant Science 20%3A 70%2D71&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Olsen O-A, Perroud P-F, Johansen W, Demko V (2015) DEK1; missing piece in puzzle of plant development. Trends in Plant Science 20: 70-71

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Olsen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=DEK1; missing piece in puzzle of plant development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=DEK1; missing piece in puzzle of plant development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Olsen&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Perroud P%2DF%2C Cove DJ%2C Quatrano RS%2C McDaniel SF %282011%29 An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines%2E New Phytologist 191%3A 301%2D306&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Perroud P-F, Cove DJ, Quatrano RS, McDaniel SF (2011) An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines. New Phytologist 191: 301-306

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Perroud&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Perroud&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Perroud P%2DF%2C Demko V%2C Johansen W%2C Wilson RC%2C Olsen O%2DA%2C Quatrano RS %282014%29 Defective Kernel 1 %28DEK1%29 is required for three%2Ddimensional growth in Physcomitrella patens%2E New Phytologist 203%3A 794%2D804&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Perroud P-F, Demko V, Johansen W, Wilson RC, Olsen O-A, Quatrano RS (2014) Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens. New Phytologist 203: 794-804

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Perroud&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Perroud&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Pfaffl MW %282001%29 A new mathematical model for relative quantification in real%2Dtime RT%2DPCR%2E Nucleic Acids Research 29&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pfaffl&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A new mathematical model for relative quantification in real-time RT-PCR.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A new mathematical model for relative quantification in real-time RT-PCR.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pfaffl&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Pires ND%2C Dolan L %282012%29 Morphological evolution in land plants%3A new designs with old genes%2E Philosophical Transactions of the Royal Society B%2DBiological Sciences 367%3A 508%2D518&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pires ND, Dolan L (2012) Morphological evolution in land plants: new designs with old genes. Philosophical Transactions of the Royal Society B-Biological Sciences 367: 508-518

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pires&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Morphological evolution in land plants: new designs with old genes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Morphological evolution in land plants: new designs with old genes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pires&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1


Prigge MJ, Bezanilla M (2010) Evolutionary crossroads in developmental biology: Physcomitrella patens. Development 137: 3535-3543


Rudenko G, Hohenester E, Muller YA (2001) LG/LNS domains: multiple functions - one business end? Trends in BiochemicalSciences 26: 363-368


Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM (2009) Amplification efficiency:linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Research 37


Saunders MJ, Hepler PK (1982) Calcium ionophore a23187 stimulates cytokinin-like mitosis in funaria. Science 217: 943-945Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schaefer DG, Delacote F, Charlot F, Vrielynck N, Guyon-Debast A, Le Guin S, Neuhaus JM, Doutriaux MP, Nogue F (2010) RAD51loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens. DNA Repair9: 526-533


Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant Journal 11: 1195-1206Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shinkai-Ouchi F, Koyama S, Ono Y, Hata S, Ojima K, Shindo M, duVerle D, Ueno M, Kitamura F, Doi N, Takigawa I, Mamitsuka H,Sorimachi H (2016) Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis UsingNewly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array. Molecular & Cellular Proteomics 15: 1262-1280


Sorimachi H, Mamitsuka H, Ono Y (2012) Understanding the substrate specificity of conventional calpains. Biological Chemistry393: 853-871


Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bourenkow G, Bartunik H,Suzuki K, Bode W (2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism foractivation by calcium. Proceedings of the National Academy of Sciences of the United States of America 97: 588-592


Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, Friedrich P (2004) On the sequential determinantsof calpain cleavage. Journal of Biological Chemistry 279: 20775-20785


Trouiller B, Schaefer DG, Charlot F, Nogue F (2006) MSH2 is essential for the preservation of genome integrity and preventshomeologous recombination in the moss Physcomitrella patens. Nucleic Acids Research 34: 232-242


Wang CX, Barry JK, Min Z, Tordsen G, Rao AG, Olsen OA (2003) The calpain domain of the maize DEK1 protein contains theconserved catalytic triad and functions as a cysteine proteinase. Journal of Biological Chemistry 278: 34467-34474


Wendt A, Thompson VF, Goll DE (2004) Interaction of calpastatin with calpain: a review. Biological Chemistry 385: 465-472Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xiao L, Wang H, Wan P, Kuang T, He Y (2011) Genome-wide transcriptome analysis of gametophyte development in Physcomitrella www.plantphysiol.orgon February 13, 2018 - Published by Downloaded from

Copyright © 2016 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=Prigge MJ%2C Bezanilla M %282010%29 Evolutionary crossroads in developmental biology%3A Physcomitrella patens%2E Development 137%3A 3535%2D3543&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Prigge MJ, Bezanilla M (2010) Evolutionary crossroads in developmental biology: Physcomitrella patens. Development 137: 3535-3543

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Prigge&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Evolutionary crossroads in developmental biology: Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Evolutionary crossroads in developmental biology: Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Prigge&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Rudenko G%2C Hohenester E%2C Muller YA %282001%29 LG%2FLNS domains%3A multiple functions %2D one business end%3F Trends in Biochemical Sciences 26%3A 363%2D368&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Rudenko G, Hohenester E, Muller YA (2001) LG/LNS domains: multiple functions - one business end? Trends in Biochemical Sciences 26: 363-368

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rudenko&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=LG/LNS domains: multiple functions - one business end&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=LG/LNS domains: multiple functions - one business end&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rudenko&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ruijter JM%2C Ramakers C%2C Hoogaars WMH%2C Karlen Y%2C Bakker O%2C van den Hoff MJB%2C Moorman AFM %282009%29 Amplification efficiency%3A linking baseline and bias in the analysis of quantitative PCR data%2E Nucleic Acids Research 37&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Research 37

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ruijter&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ruijter&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Saunders MJ%2C Hepler PK %281982%29 Calcium ionophore a23187 stimulates cytokinin%2Dlike mitosis in funaria%2E Science 217%3A 943%2D945&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Saunders MJ, Hepler PK (1982) Calcium ionophore a23187 stimulates cytokinin-like mitosis in funaria. Science 217: 943-945

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Saunders&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Calcium ionophore a23187 stimulates cytokinin-like mitosis in funaria&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Calcium ionophore a23187 stimulates cytokinin-like mitosis in funaria&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Saunders&as_ylo=1982&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schaefer DG%2C Delacote F%2C Charlot F%2C Vrielynck N%2C Guyon%2DDebast A%2C Le Guin S%2C Neuhaus JM%2C Doutriaux MP%2C Nogue F %282010%29 RAD51 loss of function abolishes gene targeting and de%2Drepresses illegitimate integration in the moss Physcomitrella patens%2E DNA Repair 9%3A 526%2D533&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Schaefer DG, Delacote F, Charlot F, Vrielynck N, Guyon-Debast A, Le Guin S, Neuhaus JM, Doutriaux MP, Nogue F (2010) RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens. DNA Repair 9: 526-533

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schaefer&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schaefer&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schaefer DG%2C Zryd JP %281997%29 Efficient gene targeting in the moss Physcomitrella patens%2E Plant Journal 11%3A 1195%2D1206&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant Journal 11: 1195-1206

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schaefer&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Efficient gene targeting in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Efficient gene targeting in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schaefer&as_ylo=1997&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Shinkai%2DOuchi F%2C Koyama S%2C Ono Y%2C Hata S%2C Ojima K%2C Shindo M%2C duVerle D%2C Ueno M%2C Kitamura F%2C Doi N%2C Takigawa I%2C Mamitsuka H%2C Sorimachi H %282016%29 Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure%2DActivity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array%2E Molecular %26 Cellular Proteomics 15%3A 1262%2D1280&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shinkai-Ouchi F, Koyama S, Ono Y, Hata S, Ojima K, Shindo M, duVerle D, Ueno M, Kitamura F, Doi N, Takigawa I, Mamitsuka H, Sorimachi H (2016) Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array. Molecular & Cellular Proteomics 15: 1262-1280

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shinkai-Ouchi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shinkai-Ouchi&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sorimachi H%2C Mamitsuka H%2C Ono Y %282012%29 Understanding the substrate specificity of conventional calpains%2E Biological Chemistry 393%3A 853%2D871&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sorimachi H, Mamitsuka H, Ono Y (2012) Understanding the substrate specificity of conventional calpains. Biological Chemistry 393: 853-871

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sorimachi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Understanding the substrate specificity of conventional calpains&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Understanding the substrate specificity of conventional calpains&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sorimachi&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Strobl S%2C Fernandez%2DCatalan C%2C Braun M%2C Huber R%2C Masumoto H%2C Nakagawa K%2C Irie A%2C Sorimachi H%2C Bourenkow G%2C Bartunik H%2C Suzuki K%2C Bode W %282000%29 The crystal structure of calcium%2Dfree human m%2Dcalpain suggests an electrostatic switch mechanism for activation by calcium%2E Proceedings of the National Academy of Sciences of the United States of America 97%3A 588%2D592&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bourenkow G, Bartunik H, Suzuki K, Bode W (2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proceedings of the National Academy of Sciences of the United States of America 97: 588-592

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Strobl&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Strobl&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Tompa P%2C Buzder%2DLantos P%2C Tantos A%2C Farkas A%2C Szilagyi A%2C Banoczi Z%2C Hudecz F%2C Friedrich P %282004%29 On the sequential determinants of calpain cleavage%2E Journal of Biological Chemistry 279%3A 20775%2D20785&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, Friedrich P (2004) On the sequential determinants of calpain cleavage. Journal of Biological Chemistry 279: 20775-20785

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tompa&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=On the sequential determinants of calpain cleavage&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=On the sequential determinants of calpain cleavage&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tompa&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Trouiller B%2C Schaefer DG%2C Charlot F%2C Nogue F %282006%29 MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens%2E Nucleic Acids Research 34%3A 232%2D242&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Trouiller B, Schaefer DG, Charlot F, Nogue F (2006) MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens. Nucleic Acids Research 34: 232-242

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Trouiller&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Trouiller&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wang CX%2C Barry JK%2C Min Z%2C Tordsen G%2C Rao AG%2C Olsen OA %282003%29 The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase%2E Journal of Biological Chemistry 278%3A 34467%2D34474&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wang CX, Barry JK, Min Z, Tordsen G, Rao AG, Olsen OA (2003) The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase. Journal of Biological Chemistry 278: 34467-34474

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wendt A%2C Thompson VF%2C Goll DE %282004%29 Interaction of calpastatin with calpain%3A a review%2E Biological Chemistry 385%3A 465%2D472&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wendt A, Thompson VF, Goll DE (2004) Interaction of calpastatin with calpain: a review. Biological Chemistry 385: 465-472

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wendt&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Interaction of calpastatin with calpain: a review&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Interaction of calpastatin with calpain: a review&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wendt&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1


patens. Bmc Plant Biology 11Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yan N (2013) Structural advances for the major facilitator superfamily (MFS) transporters. Trends in Biochemical Sciences 38: 151-159


Zhao S, Liang Z, Demko V, Wilson R, Johansen W, Olsen O-A, Shalchian-Tabrizi K (2012) Massive expansion of the calpain genefamily in unicellular eukaryotes. Bmc Evolutionary Biology 12



http://www.ncbi.nlm.nih.gov/pubmed?term=Xiao L%2C Wang H%2C Wan P%2C Kuang T%2C He Y %282011%29 Genome%2Dwide transcriptome analysis of gametophyte development in Physcomitrella patens%2E Bmc Plant Biology 11&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Xiao L, Wang H, Wan P, Kuang T, He Y (2011) Genome-wide transcriptome analysis of gametophyte development in Physcomitrella patens. Bmc Plant Biology 11

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xiao&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Genome-wide transcriptome analysis of gametophyte development in Physcomitrella patens.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Genome-wide transcriptome analysis of gametophyte development in Physcomitrella patens.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xiao&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Yan N %282013%29 Structural advances for the major facilitator superfamily %28MFS%29 transporters%2E Trends in Biochemical Sciences 38%3A 151%2D159&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Yan N (2013) Structural advances for the major facilitator superfamily (MFS) transporters. Trends in Biochemical Sciences 38: 151-159

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yan&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Structural advances for the major facilitator superfamily (MFS) transporters&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Structural advances for the major facilitator superfamily (MFS) transporters&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yan&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhao S%2C Liang Z%2C Demko V%2C Wilson R%2C Johansen W%2C Olsen O%2DA%2C Shalchian%2DTabrizi K %282012%29 Massive expansion of the calpain gene family in unicellular eukaryotes%2E Bmc Evolutionary Biology 12&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhao S, Liang Z, Demko V, Wilson R, Johansen W, Olsen O-A, Shalchian-Tabrizi K (2012) Massive expansion of the calpain gene family in unicellular eukaryotes. Bmc Evolutionary Biology 12

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhao&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Massive expansion of the calpain gene family in unicellular eukaryotes.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Massive expansion of the calpain gene family in unicellular eukaryotes.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1


Documents

The DEK1 calpain Linker functions in three-dimensional body