Supporting Information legends
Supporting Table S1. An overview of all TML-calpains identified in eukaryotes using Blast
searches.
Supporting Table S2. The sequences of all oligonucleotides used in this work.
Supporting Data S1. The coding sequences (CDS) of the two DEK1 Marchantia
polymorpha genes (A and B) used in this study. Annotation of the CDS was performed using
the FGENESH++ algorithm.
Supporting Data S2. Original data set from the CAPS coevolution analysis of plant DEK1
and animal CAPN2 CysPc-C2L domains. Sheet 1 contains a list of all coevolution groups and
coevolving residues within CysPc-C2L domains of CAPN2 orthologs. Sheet 2 contains a list
of all coevolution groups and coevolving residues within CysPc-C2L domains of DEK1
orthologs.
Supporting Data S3. The protein alignments used to construct the phylogenetic trees in
Figure 1 and Supporting Figure S2. The protein alignments are given in Nexus format.
Supporting Figure S1. Domain structure of TML-calpains. The length of the bars is
proportional to the number of amino acids in the polypeptide chains. CysPc, calpain-like thiol
protease catalytic domain; C2L, C2-like domain; TMs, transmembrane motifs; LG3,
Laminin_G_3 domain; Nter, N-terminal anchor helix; PEF, penta-EF-hand; TPRs,
tetratricopeptide repeat; WW, domain with two highly conserved tryptophans; C2, protein
kinase C conserved region 2. Te., Tetrahymena; Th., Thecamonas; Tr., Trichomonas; P.,
Phytopthora; A., Albugo; E., Ectocarpus. For comparison, a metazoan classical calpain
(Metazoa) is also shown.
Supporting Figure S2. Phylogenetic analysis of TML-calpains with animal classical calpains
as an out-group. The CysPc domain was used to construct the tree. For each node, statistical
support values (Maximum-Likelihood bootstraps inferred using PROTGAMMALG model
and Neighbor-Joining method using JTT+G model) are marked if all are more than 75 % BP
(filled circle) or more than 50 % BP (open circle). Dashes‘-’ show the support values < 50 %
BP. Scale bar indicates the number of amino acid changes per site.
Supporting Figure S3. Analysis of site-specific levels of selection in land plant DEK1
proteins. Hyphy analysis estimating dS and dN, the selective pressures of the full-length
DEK1 sequence site by site. Lines above and below zero on the x-axis show values of dS
(grey lines) and dN (blue lines), respectively. dN/dS < 1 indicate negative selection. At the
individual codon level, 1354 codons were found to be under negative selection without a
single positively selected residue. The numbering on the x-axis corresponds to the alignment
position.
Supporting Figure S4. Comparison of the networks of coevolving amino acid residues
within the orthologs of DEK1 CysPc-C2L (a) and animal CAPN2 CysPc-C2L (b). Amino
acid residues in the networks are numbered according to Arabidopsis thaliana DEK1 (a) and
Rattus norvegicus CAPN2 (b). Protein domains to which the nodes (residues) belong are
colour-coded. Nodes are connected through edges according to the nature of their coevolution
(colored lines).
Supporting Figure S5. Western blot analysis of immunoprecipitated GFP-fusion proteins.
Proteins extracted from transgenic dek1-3/+ plants expressing: (a) MvCysPc-C2L-GFP; (a)
animal CysPc/Arabidopsis C2L-GFP; (c) mutated variants of AtCysPc-C2L-GFP. Mutations
were introduced by amino acid substitutions in conserved Ca2+ binding sites. m.v2 = rat mcalpain CysPc/Arabidopsis C2L-GFP version2, m.v1 = rat m-calpain CysPc/Arabidopsis
C2L-GFP version1, µ.v2 = rat µ-calpain CysPc/Arabidopsis C2L-GFP version2, µ.v1 = rat µcalpain
CysPc/Arabidopsis
C2L-GFP
version1,
p94.v2
=
human
p94
calpain
CysPc/Arabidopsis C2L-GFP version2, p94.v1 = human p94 calpain CysPc/Arabidopsis C2LGFP version1.
Supporting Figure S6. Scheme showing the three chimeric animal/plant CysPc-C2L
sequence compositions. Using the pRPS5A:AtCysPc-C2L-GFP complementation construct as
template, the CysPc-encoding sequence was replaced by the rat or human CysPc-encoding
region. Two different versions (v.1 and v.2) of each chimera (animal CysPc/Arabidopsis C2L)
were made, differing in the start point of the sequence encoding the N-terminal region of
CysPc. Amino acids shaded in grey are animal CysPc sequences. The numbering corresponds
to the amino acid in the respective protein sequences. The template sequences used in the de
novo synthesis of the chimera constructs were: rat Capn1, NM_019152.2; rat Capn2,
NM_017116.2; human Capn3, NM_000070.2; Arabidopsis thaliana DEK1, NM_001198309.
The DNA sequence corresponding to the amino acid sequence D268GTN….ERPTR315 in p94
is the IS1 sequence and was omitted from the chimera constructs. Rn, Rattus norvegicus; Hs,
Homo sapiens; At, Arabidopsis thaliana.
Supporting Figure S7. Analyses of transgenic dek1-3/+ plants expressing the mutant variants
of the pRPS5A:AtCysPc-C2L-GFP constructs. (a) RT-PCR experiments showing the
expression of CysPc-C2L-GFP (upper panel) and actin (lower panel) transcripts in individual
T1 plants transformed with each of the seven mutated constructs (first seven lanes); lane: 1,
D106N/E185Q/E302Q/D309N line 8; 2, D106N/E185Q line 19; 3, E302Q/D309N line 5; 4,
D106N line 11; 5, E185Q line 59; 6, E302Q line 35; 7, E302Q line 22; 7, D309N line 98. As
control, a heterozygote dek1-3 plant was used (lane 8). (b) GFP expression (left) and
differential interference contrast (right) images of D106N/E185Q/E302Q/D309N in a 6-dayold root tip from line 8 (T2). (c) T2 seeds from individual siliques harvested from
D106N/E185Q/E302Q/D309N line 8, D106N/E185Q line 19 and D106N line 11 showing the
presence of typical collapsed dek1-3 mutant seeds (white arrow). For comparison, seeds from
individual siliques harvested from a wild-type plant and a heterozygote dek1-3 plant are
shown. The scale bar is 1 mm.
Supporting Figure S8. De novo synthesized DNA fragment containing the codon-optimized
Mesostigma viride DEK1-like CysPc-C2L sequence used in the complementation assay. The
optimized cDNA sequence is shaded in grey, and introduced initiation start site (ATG) is
shown in bold. 5'-XbaI (underlined) and 3'-AscI (dotted line) restriction sites used in the
construction of the binary T-DNA vector pRPS5A:MvCysPc-C2L-GFP, are shown.