Supplementary Materials forscience.sciencemag.org/content/suppl/2014/03/19/... · . Supplementary Materials for . Contribution of NAC Transcription Factors to Plant Adaptation to

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Supplementary Materials for

Contribution of NAC Transcription Factors to Plant Adaptation to Land

Bo Xu, Misato Ohtani,* Masatoshi Yamaguchi, Kiminori Toyooka, Mayumi Wakazaki, Mayuko Sato, Minoru Kubo, Yoshimi Nakano, Ryosuke Sano, Yuji Hiwatashi, Takashi

Murata, Tetsuya Kurata, Arata Yoneda, Ko Kato, Mitsuyasu Hasebe, Taku Demura*

*Corresponding author. E-mail: [email protected] (T.D.); [email protected] (M.O.)

Published 20 March 2014 on Science Express DOI: 10.1126/science. 1248417

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S31 Tables S1 to S4, S6, and S7 References (28–45)

Other Supplementary Materials for this manuscript include the following: (available at www.sciencemag.org/cgi/content/full/science.1248417/DC1)

Tables S5 and S8 to S14

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Materials and Methods Plant culture

Wild-type P. patens ssp. patens Gransden 2004 (19) was grown on BCDAT medium containing 1 mM MgSO4, 10 mM KNO3, 45 µM FeSO4, 1.8 mM KH2PO4 (pH 6.5), trace element solution (alternative TES, 0.22 µM CuSO4, 0.19 µM ZnSO4, 10 µM H3BO4, 0.10 µM Na2MoO4, 2 µM MnCl2, 0.23 µM CoCl2, and 0.17 µM KI), 1 mM CaCl2, 5 mM diammonium(+)-tartrate, and 0.8% (w/v) agar (WAKO) at 25°C under continuous light conditions (28). To induce gametophores, protonemata were transferred onto autoclaved Jiffy-7 (Jiffy Products International AS, Kristansand, Norway) and grown at 25°C under continuous light conditions. Sporophyte formation was induced by incubation of gametophores at 15°C under 16 h dark and 8 h light cycle. The Columbia ecotype of A. thaliana was cultured on germination medium (GM) containing MS medium, 0.5% (w/v) sucrose, 0.05% (v/v) MES-KOH (pH 5.8), B5 vitamins, and 0.6% Gellan Gum (w/v, WAKO) at 22˚C under 16 h-white light and 8 h-dark conditions. Vector construction and plant transformation

To generate inducible overexpressors in A. thaliana, the coding region sequences of PpVNS1 to PpVNS8 (accession number AB898081 to AB898088, respectively) were amplified by PCR and then inserted into the ApaI/PacI sites of the pER8 vector, an estrogen receptor-based inducible overexpression system (29). Inducible P. patens overexpressors were constructed by transferring the coding region sequences of the PpVNS genes or A. thaliana VND7 into the Gateway destination vector pPGX8 (accession number AB537482) (30), a derivative of pER8. To generate a chimeric PpVNS-GUS fragment, approximately 1 kb 5’ genomic fragments immediately before the stop codons of PpVNS genes were subcloned into the plasmid pTN85 (accession number AB267707), generating in-frame fusions of each PpVNS and GUS gene, which are thought to be localized mainly in the nuclei; approximately 1 kb long 3’ genomic fragments containing the stop codons of the PpVNS genes were also inserted into pTN85 carrying the corresponding 5’ fragments (fig. S4). Deletion mutants were generated by replacing the coding regions of PpVNS4 or PpVNS1, PpVNS6, and PpVNS7 with antibiotic selection markers for G418, hygromycin, or Blasticidin S cassette carried by pTN182 (accession number AB267706), pTN186 (accession number AB542059), and p35S-loxP-BSD (accession number AB537973), respectively. Approximately 1 Kb 5’ genomic fragments immediately before the start codons of the PpVNS genes were inserted into the 5’-region of plasmids pTN182, pTN186, and p35S-loxP-BSD, respectively; approximately 1 Kb 3’ genomic fragments containing the stop codons of the PpVNS genes were subcloned into 3’-regions of these plasmids (fig. S8). To construct the triple deletion mutant ppvns1 ppvns6 ppvns7, the ppvns1 deletion construct was introduced into the deletion mutant ppvns7-1 line; the ppvns6 deletion construct was introduced into the resultant double deletion mutant ppvns1 ppvns7-12 line.

PEG-mediated protoplast transformation was performed as reported previously to generate transgenic P. patens (28). Stable transformants with targeted DNA insertions were identified using PCR. These transformants were further analyzed by southern blotting to eliminate those with unexpected insertions in the genome. Genomic DNA was extracted from protonema using the CTAB method. Enzymes for digestion and the probe used for hybridisation of 2 μg genomic DNA are shown in figures S4 and S8. Primer sequences are listed in table S11.

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None of the P. pantens PpVNS-GUS transformants, overexpressors, or deletion mutants exhibited macroscopic differences from wild-type P. patens. Thus, there is no disturbance of normal development by integration of exogenous DNA fragments into the genome in the PpVNS-GUS transformants and overexpressors. Microscopy analysis

Observation of GUS activity and sectioning of GUS-stained samples (7-µm thick) were performed as previously reported (28). The stem samples of the wild type and ppvns4 were fixed in 100 mM sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde and 2% glutaraldehyde, and then fixed in 50 mM cacodylate buffer (pH 7.4) containing 1% OsO4. In the case of sporophytes of the wild type and ppvns4, the samples were fixed in 100 mM sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde, 2% glutaraldehyde, and 0.01% triton-X100, and then fixed in 50 mM cacodylate buffer (pH 7.4) containing 1% OsO4. The fixed samples were embedded in LR-White resin. 1-µm sections were stained with 0.1% (w/v) Toluidine Blue O at 80°C for 10 sec. Observations were made using a microscope equipped with Nomarski optics (BX51-DIC, Olympus) or Nomarski optics (BX53-DIC; Olympus). For the detection of GUS signals, the gametophores were sampled at 28 days after the induction. To check their viability, 4-day-old transgenic protonemata were treated with or without 1 μM 17-β-estradiol solution, exposed to UV light for 30 min (312 nm: F4-UV-312; NIPPON Genetics), or pre-incubated at 42°C for 10 min, and then incubated in Evans Blue staining buffer (3% Evans Blue [w/v, Sigma], 50 mM NaH2PO4 [pH 7.0]) at room temperature for 3 min, followed by washing four times with deionized water. The samples were observed using a microscope equipped with Nomarski optics (BX51-DIC, Olympus). Overexpression analysis

For induced overexpression analysis, 10-day-old transgenic A. thaliana harbouring estrogen-inducible PpVNS constructs were treated with 10 μM 17-β-estradiol solution (Wako), while for P. patens overexpressors, 4-day-old protonemata and 28-day-old gametophores (after inoculation on BCDAT medium) were treated with 1 μM 17-β-estradiol solution. Quantitative RT-PCR analysis

To analyze tissue-specific expression of the PpVNS genes, protonemata 6 days after inoculation, as well as the upper parts including shoot tips and lower parts including rhizoids from 28-day-old gametophores grown on Jiffy-7, were sampled with fine tweezers. For expression analysis in P. patens overexpressors, 4-day-old protonemata were treated with 1 μM 17-β-estradiol solution, and then sampled after 12 h of treatment. For the A. thaliana overexpression lines, 10-day-old seedlings were sampled after 24 h of treatment with 10 μM 17-β-estradiol. Total RNAs were extracted, treated with DNase I, and subjected to first-strand cDNA synthesis. RT products were used as templates in a PCR reaction using a LightCycler 480 SYBR Green I Master kit (Roche). The primer sets were designed to anneal to the 3’ UTRs of PpVNS genes (primer sequences are listed in table S11). For A. thaliana genes, primer sets reported previously (2) were used. All PCR reactions were performed in a LightCycler 480 II (Roche). Quantitative RT-PCR analysis was independently carried out in triplicate, and the data were analysed with LightCycler 480 II software. Target gene abundance was normalized to the internal control gene ubiquitin 10 for the P. patens tissues (31). Assay of water transport activity

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To monitor how far the Evans Blue dye was transported via hydroids in the midribs of leaves, the 12th to 20th leaves from the shoot tips of 6-week-old wild-type and triple deletion mutant ppvns1 ppvns6 ppvns7 plants were used. We collected leaves from 3 individuals per lines, thus 27 leaves per lines were examined. To examine the water transport activity of the stem, the parts of the stem where rhizoids were attached were removed from 6-week-old wild-type and ppvns4 deletion mutant plants, and the remaining parts of the shoots were stained with Evans Blue staining buffer (3% Evans Blue [w/v, Sigma], 50 mM NaH2PO4 [pH 7.0]). Incubation was performed for 30, 60, or 90 min at room temperature, followed by washing four times with deionized water. The leaves were removed for clear observation, and the samples were observed using a microscope equipped with Nomarski optics (BX53-DIC, Olympus). Low-humidity treatment analysis

The saturated sodium chloride solution method was used to prepare the condition with 75% relative humidity at 25°C (32, 33). The 8 week-old wild-type, ppvns1 ppvns6 ppvns7 triple mutant, and ppvns4 mutant P. patens were transplanted onto BCDAT plates, and transferred into the sealed chamber with 75% relative humidity. Thirty six individuals were incubated at 25°C under continuous light condition for 8 h, and subjected to the observation to check the wilting of leaves. Observation by transmission electron microscopy

For transmission electron microscopy, the 12th leaf from each shoot tip was collected from 2 individuals for the wild type and ppvns1 ppvns6 ppvns7 line 35, and 3 individuals for ppvns1 ppvns6 ppvns7 line 8, and then fixed in 100 mM sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde and 2% glutaraldehyde, and then fixed in 50 mM cacodylate buffer (pH 7.4) containing 1% OsO4. In the case of sporophytes of the wild type and ppvns4, the samples were fixed in 100 mM sodium cacodylate buffer (pH 7.4) containing 4% paraformaldehyde, 2% glutaraldehyde, and 0.01% triton-X100, and then fixed in 50 mM cacodylate buffer (pH 7.4) containing 1% OsO4. The fixed samples were embedded in LR-White resin. Ultra-thin sections (80-nm thick) were stained with uranyl acetate solution and lead solution and observed by JEM-1400 (JEOL, Akishima, Japan). For measurement of cell wall thickness of stereid cells, the cell wall regions in which the inner edge of cell wall parallels to the outer edges of that were selected, and the distance between both edges were measured using the ImageJ software (http://rsb.info.nih.gov/ij/). Numbers of stereid cells examined in this analysis were shown in table S3. Molecular phylogenetic analysis of VNS-, MYB46-, and XCP1 cysteine peptidase-like proteins in land plants

Amino acid sequences of VNS-, MYB46-, and XCP1 cysteine peptidase-like proteins in each land plant were retrieved with query sequences of VND7, MYB46, and XCP1, respectively from public data bases as follows; Phytozome v9.1 for A. thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii, P. patens, Chlamydomonas reinhardtii, Volvox carteri, Coccomyxa subellipsoidea C-169, Micromonas pusilla RCC299, Micromonas pusilla CCMP1545, and Ostreococcus lucimarinus (34) (http://www.phytozome.net); and oneKP project for Marchantia polymorpha (http://www.bioinfodata.org/Blast4OneKP/). These amino acid sequences were aligned by MAFFT v7.130b with L-INS-I (35) (http://mafft.cbrc.jp/alignment/server/). Aligned sequences used for phylogenetic analysis

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were selected with MEGA5.22 package (36). The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model with 1,000 bootstrap replicates (37). The analysis involved 251, 312, and 158 sequences for VNS, MYB46-, and XCP1 cysteine peptidases-like proteins, respectively. All positions containing gaps and missing data were eliminated. There were a total of 128, 101 and 170 positions for VNS-, MYB46-, and XCP1 cysteine peptidases-like proteins, respectively in the final dataset (table S12 to S14). Gene expression profiling by RNA-seq

4-day-old PpVNS7 overexpressor ER-PpVNS7 protonemata were treated with 1 μM 17-β-estradiol solution, and then sampled after 12 h of treatment. Total RNAs were extracted and treated with DNase I. RNA-seq libraries were prepared using NEBNext Ultra RNA Library Prep Kit (NEB) with index oligo for each library (NEB). Multiplex-sequencing was conducted on GAIIx (illumina) and 33 bp reads were obtained. To map raw reads to moss genome sequences with annotated gene models, genome assembly (Ppatens_152.fa) and gff annotation (Ppatens_152_gene_exons.gff3) files of Physcomitrella patens subsp patens v1.6 were obtained via Phytozome website (http://www.phytozome.net; downloaded at ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Ppatens_v1.6/). After demultiplexing, 33 bp reads were aligned to the genome sequences by using TopHat v2.0.10 (38) (http://tophat.cbcb.umd.edu) with the following options: "--segment-length 16", "--max-multihits 20", "--report-secondary-alignments", "--GTF Ppatens_152_gene_exons.gff3", and the other options set as default. Transcript expression and differentially expressed (DE) genes were defined with Cuffdiff 2 program (38), a part of Cufflinks package (http://cufflinks.cbcb.umd.edu). Cuffdiff was run with default setting except "--min-alignment-count 1". Relative expression level was calculated in fragments per kilobase of exon model per million mapped reads (FPKM). Genes with q-value < 0.01 were regarded as DE genes. Raw RNA-seq data was deposited in the DDBJ Sequence Read Archive (DRA) database (DRA001341). GO analysis

For gene ontology analysis, gene lists which includes genes significantly up- or down-regulated at least 4-fold in P. patens overexpressing PpVNS7 (2910 or 3724 genes, respectively) and direct target genes of VND/NST/SND in A. thaliana (448 genes), were analyzed with BiNGO 2.44 (http://www.psb.ugent.be/cbd/papers/BiNGO/Home.html) (39) which run on Cytoscape v3.0.2 (http://www.cytoscape.org/). Hypergeometric tests with Benjamini & Hochberg False Discovery Rate (FDR) were employed with FDR < 0.05 to identify significantly overrepresented GO terms. GO annotation information for v1.6 of P. patens genome (40) was downloaded from COSMOSS website (https://www.cosmoss.org/physcome_project/wiki/Downloads).

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Supplementary text Supplementary Note 1 | Phenotypes of ppvns4 mutant in sporophyte.

Hydroid cells differentiate in the seta and foot regions of the sporophyte in some mosses (13). In P. patens, the seta does not elongate and cells located in the central region of a seta and a foot do not show characteristic features of hydroid cells (Fig. 3, I and J), such as thin cell wall and autolysis of cytoplasm (Fig. 3D).

Epidermal cells of foot regions are differentiated into transfer cells in P. patens (Fig. 3, G to J, and fig. S11). Transfer cells have extensive wall ingrowths to increase the surface area of the plasma membrane, increasing transport of water and nutrients (13, 41). In wild-type plants, transfer cells have elaborate wall ingrowths and dense cytoplasm, and were easily-distinguishable on the basis of deep staining by the Toluidine Blue dye. Thus, the boundary of transfer cell differentiation, i.e. the boundary between the seta and the foot, was well-defined (please see red arrowheads in Fig. 3H). In contrast, such a boundary was unclear in the ppvns4 mutants; the epidermal cells in the foot did not stain uniformly (Fig. 3L). Magnified transverse views showed that the wild type transfer cells have elongated plastids and a large number of small vacuoles (Fig. 3J, and fig. S11), while round-shaped plastids and malformed vacuoles were detected in ppvns4 (Fig. 3N, and fig. S11). In addition, ppvns4 had irregular shaped cells in the central regions of the seta and foot (Fig. 3, I, J, M, N, and fig. S11). These observations indicate that the ppvns4 deletion mutation affects cell differentiation in the sporophyte seta and foot. Supplementary Note 2 | Genome-wide expression profiling of P. patens overexpressing PpVNS7. Genes up- and down-regulated by PpPNS7

To dissect the molecular basis of events downstream of PpVNS genes in P. patens, we carried out genome-wide expression analysis of transgenic P. patens overexpressing PpVNS7 using RNA-sequencing. Overexpression was induced by treatment with 10 µM 17-β-estradiol. Three biological replicates of 4-day-old protonemata were harvested either just before or 12 h after the 17-β-estradiol treatment and RNA-sequencing was performed. Approximately 53 million high-quality reads for each sample were generated and mapped against the P. patens reference transcripts. 25,302 of the 32,273 (78.4%) reference transcripts were represented by at least one read, of which 8,613 and 8,571 transcripts were statistically up- or down-regulated (q-value < 0.01), respectively, by PpVNS7 overexpression (table S5). Table S6 lists top 40 transcripts with highly increased expression in the PpVNS7 overexpressor, which includes PpVNS7 showing more than 6,000-fold upregulated expression, several genes encoding possible cell wall-related enzymes such as xyloglucan endotransglucosylase hydrolase (Pp1s141_131V6), alpha expansin (Pp1s266_66V6), alpha-L-arabinofuranosidase (Pp1s317_21V6) and pectinesterase (Pp1s258_34V6), and several genes encoding putative transcription factors such as BEL1-like homeodomain protein (Pp1s115_128V6), C2 domain-containing proteins (Pp1s336_70V6 and Pp1s383_3V6), dof domain protein (Pp1s199_87V6), and AP2 domain-containing transcription factors (Pp1s31_112V6, Pp1s259_104V6, and Pp1s53_80V6). To make clear the molecular events downstream of PpVNS7 genome-

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widely, we constructed gene ontology (GO) term networks for 2,910 statistically upregulated transcripts with at least 4-fold change (excluding transcripts with no read before 17-β-estradiol treatment) (Fig. 4K, table S8, and fig. S16 to S18), in which, as expected, GO terms related to cell walls and transcription factors such as “cell wall”, “carbohydrate metabolic process”, and “transcription factor activity” were highlighted. In contrast, the list of top 40 transcripts with highly decreased expression in the PpVNS7 overexpressor (table S7) and the GO term networks for 3,724 statistically down-regulated transcripts with at least 4-fold change (excluding transcripts with no read in the PpVNS7 overexpressor) (fig. S21 to S24 and table S8) showed a higher variety of genes and GO terms, including those related to photosynthesis and chloroplast, which might reflects the degradation of chloroplasts caused by the PpVNS7 overexpression. Conservation of VNS target genes between A. thaliana and P. patens

A number of putative direct target genes of A. thaliana VND/NST/SND genes were identified through transcriptome analyses of transgenic A. thaliana plants or cultured cells overexpressing the VND/NST/SND genes (4-6). We compiled these data in one integrated list containing 448 putative target genes of A. thaliana VND/NST/SND genes (table S9), with which GO term networks were provided (Fig. 4L, table S8, and fig. S25 to S27). Comparison of the networks for A. thaliana genes with those of P. patens genes upregulated downstream of PpVNS7 identified a number of overlapping GO terms such as “catalytic activity”, “carbohydrate metabolic process”, “cell wall”, and “external encapsulating structure”, suggesting the possible conservation of gene regulatory networks downstream of VND/NST/SND genes between P. patens and A. thaliana.

To further consider the conservation of gene regulatory networks, we examined the expression changes of several sets of P. patens transcripts with possible orthologous relationship to A. thaliana genes that function in major processes, such as transcriptional regulation, cell wall formation and programmed cell death, downstream of VND/NST/SND genes (Fig. 4M, and table S10). We found 5 (Pp1s189_29V6.1, Pp1s24_48V6.1, Pp1s149_164V6.1, Pp1s277_14V6.1, and Pp1s211_139V6.1) and 3 transcripts (Pp1s433_19V6.1, Pp1s78_152V6.1, and Pp1s420_4V6.1) as putative orthologs of AtMYB46/AtMYB83/AtMYB103 and AtMYB85, respectively (table S9, and fig. S28), of which one putative AtMYB46/AtMYB83/AtMYB103 ortholog (Pp1s189_29V6) and all three putative AtMYB85 orthologs showed statistically upregulated expression after induction of PpPNS7 expression (table S10). In addition, most of the putative orthologs of KNAT7/IRX11, LBD30/ASL19, and LBD15/ASL11 (19) (http://moss.nibb.ac.jp/treedb/) are also highly expressed in P. patens after induction of PpVNS7 overexpression, even though not always being supported statistically.

A. thaliana has ten CesA genes, CesA1 to CesA10, of which three (CesA4, CesA7, and CesA8) are required in secondary cell wall formation and the direct or indirect targets of VND/NST/SND genes, while the others function in primary cell wall formation (42). Roberts and Bushoven (43) described six P. patens genes, PpCesA3 to PpCesA8, encoding putative cellulose synthases, which are thought to diverge independently of vascular plant CesAs, and therefore there is no distinct classification of CesAs for primary and secondary cell wall synthases. However, since a phylogenetic tree of CesA super family including CesA genes of P. patens, A. thaliana and O. sativa indicates that P. patens CesA genes are most closely related to the CesA genes of A. thaliana and O. sativa

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than other CESA-like genes (43), it is likely that these six P. patens CesA genes are putative orthologues of A. thaliana CesA genes. Of these putative CesA orthologues, we found that PpCesA3 (Pp1s8_137V6) shows highly upregulated expression caused by PpVNS7 overexpression (table S10).

A number of A. thaliana genes encoding glycosyltransferases, such as IRX7, IRX9, IRX10, and IRX14, which are required for the synthesis of the glucuronoxylan backbone are expressed downstream of VND/NST/SND genes (table S8). Analysis of the P. patens genome suggested nine putative orthologous genes for the A. thaliana glycosyltransferase genes (25). Of the nine putative orthologs, six exhibited statistically upregulated expression after the overexpression of PpVNS7 (table S9).

It is known that the P. patens genome contains orthologs of all the eight core lignin biosynthetic enzymes required for the biosynthesis of p-coumaryl alcohol and coniferyl alcohol (26). In A. thaliana, the expression of a number of genes for the lignin biosynthetic enzymes such as PAL1 (At2g37040), 4CL1 (At1g51680), CCoAOMT1 (At4G34050), CCR2 (At1G80820), and CAD1 (At1G72680) are upregulated downstream of VND/NST/SND genes (table S8), and, as expected, the expression of about the half of their P. patens putative orthologs (12 of 21) were statistically increased by PpVNS7 overexpression (table S10).

Several A. thaliana genes encoding hydrolytic enzymes such as papain-like cysteine peptidases, XCP1 and XCP2, and an S1-type nuclease, BFN1, are expressed in A. thaliana downstream of VND/NST/SND genes (table S9). In the P. patens genome, we found 8 homologs of A. thaliana XCP1/XCP2 by phylogenetic analysis (fig. S29 and table S10), while relationship of orthologous group among them was not supported by the bootstrap value. Comparing of amino acid sequences of cysteine peptidase between A. thaliana and P. patens, ERFININ motif for pro-domain mature protein interaction in XCP1/XCP2 was conserved in Pp1s49_32v6.1 and Pp1s285_10v6.1 (44) (fig. S30). Furthermore, all 8 homologs in P. patens contain active sites of Cys and His residues. Two putative orthologs of BFN1 (45) can be identified (table S9). In A. thaliana, AtXCP1 and AtXCP2 genes are known to be the direct targets of VND/NST/SND among total 28 genes shown in fig. S29. Likewise, 5 of 8 XCP1/XCP2-like proteins and one of 2 putative BFN1 orthologs showed statistically upregulated expression in P. patens overexpressing PpVNS7 (table S10).

To validate the RNA-seq data, qRT-PCR analysis was carried out for the P. patens putative orthologs of CesA and XCP1/XCP2 that were represented by at least one read in both samples (fig. S31, A and B). A scatter plot of relative expression values obtained by RNA-Seq and qRT-PCR revealed that a good correlation between these data was found (fig. S31C).

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Fig. S1. PpVNS gene structure. Gene structure of PpVNS1 to PpVNS8 and related genes in A. thaliana. Boxes represent exons and lines represent introns. NAC subdomain regions were shown by colored boxes. They share a similar exon/intron structure with each other.

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Fig. S2. Maximum likelihood tree of PpVNS proteins and their homologs in vascular plants.(A) Whole view. (B) Magnified view indicated by (b) in (A). AtVND7 was underlined with a red line. The subgroup including AtVND7 supported by the bootstrap value 84 was indicated as VNS group. On the scale, 0.1 represents the estimated number of substitutions per site.At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow);Pp: Physcomitrella patens (purple).

AT5G53950.1|ANAC098|CUC2 Potri.001G396300.1 Potri.001G396400.1

Potri.011G115400.1 AT3G15170.1|ANAC054|CUC1

Potri.005G255900.1 Potri.002G005800.1

AT1G76420.1|ANAC031|CUC3 Os08g40030.1

Os06g23650.1 Potri.012G001400.1

Potri.015G020000.1 AT5G61430.1|ANAC100

AT5G07680.1|ANAC079 Potri.017G086200.1

AT5G39610.1|ANAC092|ORE1 AT3G29035.1|ANAC059|NAC3

Os04g38720.1 Os02g36880.1

AT5G18270.1|ANAC087 AT3G04060.1|ANAC046

Potri.019G031600.1 Potri.013G054200.1

Os01g01470.1 Os01g29840.1

Os07g48550.1 Os03g21030.1

Os12g03050.1 Os11g03370.1

Potri.015G046800.1 Potri.012G056300.1 AT3G18400.1|ANAC058

Potri.018G003800.1 Potri.006G277000.1

AT2G24430.1|ANAC038|ANAC039 Os09g32260.1

Os03g42630.1 Potri.005G098200.1

AT1G56010.2|ANAC022|NAC1 AT3G12977.1

Os12g41680.1 Os08g10080.1 Os06g46270.1

Os02g06950.1 Potri.005G225800.1

Potri.002G037100.1 AT4G28530.1|ANAC074

Os02g56600.1 Os04g43560.1

Os10g33760.1 Os03g01870.1

Smo 71303 Pp1s288 4V6.1 Pp1s227 100V6.1

Pp1s46 208V6.1 Pp1s159 57V6.1

Pp1s327 64V6.1 Pp1s164 37V6.1

Pp1s279 85V6.1 Pp1s259 58V6.1

Mpo JPYU-4534 Mpo JPYU-4536

Os09g24560.1 Os08g33670.1

Potri.011G153300.1 Potri.001G448400.1

AT1G32770.1|ANAC012|SND1|NST3 AT2G46770.1|ANAC043|NST1

Potri.014G104800.1 Potri.002G178700.1

AT3G61910.1|ANAC066|NST2 Os08g02300.1

Os06g04090.1 AT1G79580.1|ANAC033|SMB

Potri.010G176600.1 Potri.008G080000.1

Os06g33940.1 Os02g15340.1

Potri.013G092400.1 Potri.019G066000.1

AT4G10350.1|ANAC070|BRN2 AT1G33280.1|ANAC015|BRN1

Potri.007G014400.1 AT4G36160.1|ANAC076|VND2 Potri.005G116800.1

AT2G18060.1|ANAC037|VND1 Os03g03540.1

Os10g38834.1 AT5G66300.1|ANAC105|VND3

Os08g01330.1 Os04g59470.1

Potri.019G083600.1 Potri.013G113100.1

AT1G71930.1|ANAC030|VND7 Potri.001G120000.1

Os02g42970.1 Potri.003G113000.1

AT1G62700.1|ANAC026|VND5 AT1G12260.1|ANAC007|VND4

Os06g01480.1 Potri.012G126500.1

Potri.015G127400.1 AT5G62380.1|ANAC101|VND6

Smo 36139 Smo 74950

Pp1s223 12V6.1|PpVNS5 Pp1s161 73V6.1|PpVNS2




Smo 71404 Smo 89986

Mpo JPYU-9533

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92

77

62

99

64

96

28

3

55

86

87

1

0

48

71

26

95

62

90

83

96

67

89

76

84

89

90

50

40

75

97

62

22

32

90

16

70

58

26

50

41

13

15

17

32

55

8

49

30

20

54

7

63

43

21

23

35

7

30

0.1

AT5G53950.1|ANAC098|CUC2 Potri.001G396300.1 Potri.001G396400.1

Potri.011G115400.1 AT3G15170.1|ANAC054|CUC1

Potri.005G255900.1 Potri.002G005800.1

AT1G76420.1|ANAC031|CUC3 Os08g40030.1

Os06g23650.1 Potri.012G001400.1

Potri.015G020000.1 AT5G61430.1|ANAC100

AT5G07680.1|ANAC079 Potri.017G086200.1

AT5G39610.1|ANAC092|ORE1 AT3G29035.1|ANAC059|NAC3

Os04g38720.1 Os02g36880.1

AT5G18270.1|ANAC087 AT3G04060.1|ANAC046

Potri.019G031600.1 Potri.013G054200.1

Os01g01470.1 Os01g29840.1

Os07g48550.1 Os03g21030.1

Os12g03050.1 Os11g03370.1

Potri.015G046800.1 Potri.012G056300.1 AT3G18400.1|ANAC058

Potri.018G003800.1 Potri.006G277000.1

AT2G24430.1|ANAC038|ANAC039 Os09g32260.1

Os03g42630.1 Potri.005G098200.1

AT1G56010.2|ANAC022|NAC1 AT3G12977.1

Os12g41680.1 Os08g10080.1 Os06g46270.1

Os02g06950.1 Potri.005G225800.1

Potri.002G037100.1 AT4G28530.1|ANAC074

Os02g56600.1 Os04g43560.1

Os10g33760.1 Os03g01870.1

Smo 71303 Pp1s288 4V6.1 Pp1s227 100V6.1

Pp1s46 208V6.1 Pp1s159 57V6.1

Pp1s327 64V6.1 Pp1s164 37V6.1

Pp1s279 85V6.1 Pp1s259 58V6.1


Os09g24560.1 Os08g33670.1

Potri.011G153300.1 Potri.001G448400.1

AT1G32770.1|ANAC012|SND1|NST3 AT2G46770.1|ANAC043|NST1

Potri.014G104800.1 Potri.002G178700.1

AT3G61910.1|ANAC066|NST2 Os08g02300.1

Os06g04090.1 AT1G79580.1|ANAC033|SMB

Potri.010G176600.1 Potri.008G080000.1

Os06g33940.1 Os02g15340.1

Potri.013G092400.1 Potri.019G066000.1

AT4G10350.1|ANAC070|BRN2 AT1G33280.1|ANAC015|BRN1

Potri.007G014400.1 AT4G36160.1|ANAC076|VND2 Potri.005G116800.1

AT2G18060.1|ANAC037|VND1 Os03g03540.1

Os10g38834.1 AT5G66300.1|ANAC105|VND3

Os08g01330.1 Os04g59470.1

Potri.019G083600.1 Potri.013G113100.1

AT1G71930.1|ANAC030|VND7 Potri.001G120000.1

Os02g42970.1 Potri.003G113000.1

AT1G62700.1|ANAC026|VND5 AT1G12260.1|ANAC007|VND4

Os06g01480.1 Potri.012G126500.1

Potri.015G127400.1 AT5G62380.1|ANAC101|VND6

Smo 36139 Smo 74950





Smo 71404 Smo 89986

Mpo JPYU-9533 Potri.008G031800.1

Potri.010G229900.1 AT5G04410.1|ANAC078|NAC2

AT3G10500.1|ANC053 Potri.010G229700.1

AT3G10480.1|ANAC050 AT3G10490.1|ANAC051|ANAC052

Os08g44820.1 Os02g57650.1

Potri.012G038100.1 AT3G17730.1|ANAC057

Potri.015G030200.1 Os09g38000.1

Os09g38010.1 AT3G03200.1|ANAC045

AT5G17260.1|ANAC086 Potri.010G174600.1

Potri.008G081500.1 AT1G54330.1|ANAC020

Potri.017G139500.1 Potri.004G081000.1

AT1G65910.1|ANAC028 Os03g02800.1

Potri.003G089800.1 Potri.001G144400.1

Os10g42130.1 AT1G32510.1|ANAC011

AT4G17980.1|ANAC071 AT5G46590.1|ANAC096

Potri.005G058900.1 AT5G09330.1|ANAC082|VNI1

AT5G64060.1|ANAC103 Os05g35170.1

AT3G44290.1|ANAC060 AT5G22290.1|ANAC089

Os01g15640.1 Potri.001G452700.1

Potri.011G149300.1 AT4G35580.1|NTL9

AT1G33060.1|ANAC014 Potri.012G007500.1

AT5G24590.2|ANAC091|TIP AT3G49530.1|ANAC062|NTL6

Os08g06140.1 Os06g01230.1

AT1G34180.1|ANAC016 AT1G34190.1|ANAC017

Pp1s8 36V6.1 Pp1s73 182V6.1

Pp1s10 90V6.1 Pp1s44 300V6.1


Pp1s251 11V6.1 Os12g29330.1

Os11g31330.1 Os08g42400.1

AT2G33480.1|ANAC041 AT5G13180.1|ANAC083|VNI2

Os05g34310.1 AT3G04070.1|ANAC047

Potri.013G054000.1 Potri.019G031400.1

Potri.011G046700.1 Potri.004G038000.1

AT1G61110.1|ANAC025 Potri.001G404400.1


AT1G52880.1|ANAC018|ATNAM Os07g37920.1

Potri.008G089000.1 AT1G69490.1|ANAC029|NAP

Potri.006G129400.1 Os12g03040.1 Os11g03300.1

Os03g21060.1 Os01g01430.1

Os07g48450.1 Potri.001G404100.1

Potri.011G123300.1 AT4G27410.2|ANAC072|RD26

AT3G15500.1|ANAC055|NAC3 AT1G52890.1|ANAC019

Potri.002G081000.1 Potri.005G180200.1

AT1G01720.1|ANAC002|ATAF1 AT1G77450.1|ANAC032

Os01g66120.1 Os05g34830.1


Os11g08210.1 Os01g60020.1

Os03g60080.1 Os07g12340.1

Smo 105213 Pp1s259 83V6.1 Pp1s164 25V6.1

Pp1s117 16V6.1 Pp1s15 200V6.1

Pp1s140 93V6.1 Pp1s5 359V6.1

Pp1s164 33V6.1 Pp1s259 67V6.1

Mpo JPYU-6411 Smo 47266

Smo 147012 Smo 71307

Smo 69131 Potri.017G082000.1 AT5G39820.1|ANAC094

AT1G26870.1|ANAC009|FEZ Os08g33910.1

Potri.003G149700.1 Potri.007G066300.1

AT2G43000.1|ANAC042 AT3G12910.1

Os12g43530.1 Os03g56580.1

Os07g04560.1 Smo 25673

Smo 67677 AT2G17040.1|ANAC036

Os03g04070.1 Os06g51070.1

AT2G02450.1|ANAC035|LOV1 Os08g02160.1

Os05g34600.1 Os01g66490.1

Os01g64310.1 Os05g37080.1 Os12g05990.1

Os11g05614.1 Pp1s4 283V6.1

99

94

100

86

100

96

97

74

92

100

99

88

72

88

100

99

80

100

48

99

42

97

84

38

80

71

64

94

99

66

9981

61

100

96

44

99

97

87

45

99

88

80

92

93

60

73

37

79

92

25

51

6315

91

51

80

93

94

82

28

92

28

24

53

80

41

74

71

90

96

99

26

71

99

87

30

19

90

97

84

97

99

81

85

44

25

71

98

37

100

71

38

99

89

66

51

45

45

16

62

75

31

30

50

48

90

21

4689

96

42

81

98

55

4

24

62

21

91

40

48

95

34

20

99

18

54

47

99

29

48

68

88

29

12

66

90

92

77

62

99

55

64

96

41

5

31

30

10

89

14

8

12

28

34

13

3

55

82

86

87

1

16

86

64

90

14

52

52

29

0

58

3

91

90

48

71

26

95

62

90

83

96

67

50

17

60

8

43

87

89

76

84

23

25

46

35

19

89

59

3

9

10

15

48

90

50

40

75

97

62

22

32

90

16

70

58

26

50

41

13

15

17

32

55

8

49

30

20

54

7

63

43

21

23

35

7

30

0.1

A

(b)

(c)

B

VNS group

11

Fig. S2. (cont.)(C) Magnified view indicated by (c) in (A). On the scale, 0.1 represents the estimated number of substitutions per site. At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow); Pp: Physcomitrella patens (purple).

Potri.008G031800.1 Potri.010G229900.1

AT5G04410.1|ANAC078|NAC2 AT3G10500.1|ANC053 Potri.010G229700.1

AT3G10480.1|ANAC050 AT3G10490.1|ANAC051|ANAC052

Os08g44820.1 Os02g57650.1

Potri.012G038100.1 AT3G17730.1|ANAC057

Potri.015G030200.1 Os09g38000.1

Os09g38010.1 AT3G03200.1|ANAC045

AT5G17260.1|ANAC086 Potri.010G174600.1

Potri.008G081500.1 AT1G54330.1|ANAC020

Potri.017G139500.1 Potri.004G081000.1

AT1G65910.1|ANAC028 Os03g02800.1

Potri.003G089800.1 Potri.001G144400.1

Os10g42130.1 AT1G32510.1|ANAC011

AT4G17980.1|ANAC071 AT5G46590.1|ANAC096

Potri.005G058900.1 AT5G09330.1|ANAC082|VNI1

AT5G64060.1|ANAC103 Os05g35170.1

AT3G44290.1|ANAC060 AT5G22290.1|ANAC089

Os01g15640.1 Potri.001G452700.1

Potri.011G149300.1 AT4G35580.1|NTL9

AT1G33060.1|ANAC014 Potri.012G007500.1

AT5G24590.2|ANAC091|TIP AT3G49530.1|ANAC062|NTL6

Os08g06140.1 Os06g01230.1

AT1G34180.1|ANAC016 AT1G34190.1|ANAC017

Pp1s8 36V6.1 Pp1s73 182V6.1

Pp1s10 90V6.1 Pp1s44 300V6.1


Pp1s251 11V6.1 Os12g29330.1

Os11g31330.1 Os08g42400.1

AT2G33480.1|ANAC041 AT5G13180.1|ANAC083|VNI2

Os05g34310.1 AT3G04070.1|ANAC047

Potri.013G054000.1 Potri.019G031400.1

Potri.011G046700.1 Potri.004G038000.1

AT1G61110.1|ANAC025 Potri.001G404400.1


AT1G52880.1|ANAC018|ATNAM Os07g37920.1

Potri.008G089000.1 AT1G69490.1|ANAC029|NAP

Potri.006G129400.1 Os12g03040.1 Os11g03300.1

Os03g21060.1 Os01g01430.1

Os07g48450.1 Potri.001G404100.1

Potri.011G123300.1 AT4G27410.2|ANAC072|RD26

AT3G15500.1|ANAC055|NAC3 AT1G52890.1|ANAC019

Potri.002G081000.1 Potri.005G180200.1


Os01g66120.1 Os05g34830.1


Os11g08210.1 Os01g60020.1

Os03g60080.1 Os07g12340.1

Smo 105213 Pp1s259 83V6.1 Pp1s164 25V6.1

Pp1s117 16V6.1 Pp1s15 200V6.1

Pp1s140 93V6.1 Pp1s5 359V6.1

Pp1s164 33V6.1 Pp1s259 67V6.1


Smo 147012 Smo 71307

Smo 69131 Potri.017G082000.1 AT5G39820.1|ANAC094

AT1G26870.1|ANAC009|FEZ Os08g33910.1

Potri.003G149700.1 Potri.007G066300.1

AT2G43000.1|ANAC042 AT3G12910.1

Os12g43530.1 Os03g56580.1

Os07g04560.1 Smo 25673

Smo 67677 AT2G17040.1|ANAC036

Os03g04070.1 Os06g51070.1

AT2G02450.1|ANAC035|LOV1 Os08g02160.1

Os05g34600.1 Os01g66490.1

Os01g64310.1 Os05g37080.1 Os12g05990.1

Os11g05614.1 Pp1s4 283V6.1

99

94

100

86

100

96

97

74

100

99

88

72

88

99

80

100

48

99

42

38

80

71

64

94

99

9981

61

96

44

99

87

45

99

88

80

92

93

60

90

96

26

71

99

87

19

84

99

81

85

44

25

98

37

100

71

38

99

89

45

31

30

50

48

90

4689

81

98

55

24

62

21

91

40

48

95

34

20

99

18

99

29

48

68

88

29

55

41

5

31

30

10

89

14

8

12

34

13

82

16

86

64

90

14

52

52

29

58

3

91

90

50

17

60

8

43

87

23

25

46

35

19

59

3

9

10

15

48

0.1

C

12

Fig. S3. Expression of PpVNS genes in P. patens. (A) Tissues used for qRT-PCR analysis. Total RNAs were extracted from protonemata (p) and from the upper-part (u), including shoot tip, and lower-part (l), including rhizoids, of gametophores. (B) Relative abundance of PpVNS1-8 transcripts (n=3). Results are mean ± s.d.. (C to V) GUS expression patterns in gametophores and protonemata of transgenic lines PpVNS2-GUS (C to G), PpVNS5-GUS (H to L), PpVNS8-GUS (M to Q), and PpVNS3-GUS (R to V): newly emerged leaves (D, I, N, and S), leaves (E, J, O, and T), rhizoids (F, K, P, and U, yellow arrows), and protonemata (G, L, Q, and V). No GUS expression was observed in the PpVNS3-GUS lines (R to V). Scale bars, 5 mm (C, H, M, and R) or 100 µm (D to G, I to L, N to Q, S to V).

13

Fig. S4. Generation of PpVNS-GUS transformants. (A) Schematic construction of PpVNS-GUS fusion genes. Boxes and lines between boxes indicate exons and introns, respectively. Blue and orange arrows indicate the GUS gene and the nptII expression cassette, respectively. The vector-derived BamHI and BglII sites were located at the downstream of nptII gene cassette, resulting in shorter sizes of expected band (SEB) in PpVNS2 and PpVNS8 transformants than in wild type (WT). (B to I) Screening of transgenic plants with targeted insertion using Southern blotting. Genomic DNA was digested by BamHI (B and C), BglII (D, E, H, and I), or EcoRI (F and G). The digestion sites were outside of the location that was recognized by the probe, as indicated in (A) with a red box. The SEB for the transformants (upper) and for WT (lower) visualized by Southern blotting are indicated in (B to I).

14

Fig. S5. Expression of PpVNS genes in differently-aged leaves. GUS expression patterns in differently-aged leaves of 28 days of transgenic plants PpVNS1-GUS (A to D), PpVNS2-GUS (E to J), PpVNS4-GUS (K to P), PpVNS5-GUS (Q to V), PpVNS6-GUS (W to BB), PpVNS7-GUS (CC to GG), and PpVNS8-GUS (HH to MM). L3 to L18 represented positions of leaves from the shoot tip. Yellow arrows indicated GUS signals in the midrib regions. Regions indicated by black squares in (A, B, C, GG, and MM) were magnified in fig. S6. Scale bars, 200 µm.

15

Fig. S6. Expression of PpVNS genes in differently-aged leaves. Close-up views of the regions indicated by black squares in fig. S5. PpVNS1-GUS (A to E), PpVNS7-GUS (F), and PpVNS8-GUS (G) were shown. Yellow arrows indicated GUS signals in the midrib regions. Scale bars, 100 µm (A, D, E, and G) or 50 µm (B, C, and F).

16

Fig. S7. Sections of midrib regions of PpVNS-GUS leaves. Technovit sections of GUS-stained leaf samples of PpVNS-GUS plants. Close-up views of midrib regions were shown. GUS signals were detected in developing hydroid cells, stereid cells, and epidermal cells. Asterisks indicate hydroid cells. Scale bar, 20 µm.

17

Fig. S8. Construction of single or triple deletion mutants. (A) Schematic construction of deletion mutants. Boxes and lines between boxes indicate exons and introns, respectively. Blue box indicates cauliflower mosaic virus 35S promoter and green arrow indicates the nptII expression cassette. (B and C) Southern blotting analysis of transgenic plants with targeted knock-out. Genomic DNA was digested with the restriction enzyme indicated under each picture. The digestion sites were outside of the location that was recognized by the probe, as indicated in (A) with a red box. The size of expected band (SEB) visualized by Southern blotting is indicated in (B and C).

18

Fig. S9. Effects of prolonged incubation time on Evans Blue dye transport in deletion mutants. (A and B) Transport of Evans Blue dye in the leaves of wild-type (WT), ppvns1 ppvns6 ppvns7 line 8 and line 35 when the samples were incubated in dye solution for 30 min (A; calculated with samples used in the experiment showed in Fig. 2, A to C) or for 30, 60, and 90 min (B). Ratio of the distance of Evans Blue transport through the midrib, to the length of leaf, is shown. Results are mean ± s.d.; *P < 0.05 and **P < 0.01 (Welch’s t-test). Although transport of the dye increased slightly in a time-dependent manner in WT, no difference was observed in ppvns1 ppvns6 ppvns7 mutant lines. (C) Transport of Evans Blue dye through stems of WT and ppvns4 deletion mutants when the samples were incubated in dye solution for 90 min. Arrowheads indicate positions where Evans Blue dye was transported from the base. No enhancement of the dye transport was observed even after 90 min incubation in ppvns4 mutant lines. Scale bar, 500 µm.

19

Fig. S10. Phenotypes of stereid cell in triple deletion mutants. (A and B) Cell wall thickening of stereid cells of leaves in WT and ppvns1 ppvns6 ppvns7 line 8 and line 35. (A) Results from TEM images shown in Fig. 2, D to I. The numbers of examined cells were described in table S4. (B) Ten individuals were subjected to test the biological reproducibility. Five stereid cells were randomly selected from one individual of each line. Results are shown as mean ± s.d.. Reduced cell wall thickness in the triple mutants was statistically supported (**P < 0.01; Welch’s t-test). (C) Numbers of stereid cells of middle part of leaves in wild-type (WT) and ppvns1 ppvns6 ppvns7 line 8 and line 35. Results are mean ± s.d. (n=10). No difference in the number of stereid cells was observed. (D) Sizes of stereid cells of middle part of leaves in WT and ppvns1 ppvns6 ppvns7 line 8 and line 35. The size of WT was set as 1.0. Results are mean ± s.d. (n=10). The size of stereid cells of ppvns1 ppvns6 ppvns7 lines was larger than that of WT. *P < 0.05 and **P < 0.01 (Welch’s t-test).

20

Fig. S11. Defects in foot cell differentiation in ppvns4 sporophyte. Images of TEM transverse sections of a foot region of wild type (WT; A to D) and ppvns4 deletion mutant (E to H). Magnified views of cells in central regions (B and F) and transfer cells (C and G) were shown. (D and H) Close-up images of transfer cells. In ppvns4, irregular shaped-vacuoles (G) and plastid (H) were observed, indicating the deficiency of transfer cell differentiation in ppvns4. t, transfer cell; n, nucleus; m, mitochondrion; p, plastid; v, vacuole; cwi, cell wall ingrowth. For the details, please see supplementary note 1. Scale bars, 20 µm (A, E); 5 µm (B, C, F, and G); 1 µm (D); and 500 nm (H).

21

Fig. S12. Semi-qRT-PCR of estrogen-induced overexpression of PpVNS genes or AtVND7 in P. patens overexpressors. Four days after subculture, protonemata of wild type (WT) and PpVNS overexpressors were treated with 1 µM 17-β-estradiol (ER) for 8 h, and the abundance of target genes induced by the ER treatment was investigated by semi-qRT-PCR with primer sets to amplify the coding regions of the PpVNS genes or AtVND7. The ubiquitin 10 gene was used as the internal control. The PCR cycle numbers are indicated on the right. The overexpressor lines with numbers indicated by red color were used for the experiments shown in Fig. 4 and fig. S13.

22

Fig. S13. VNS proteins can induce cell death in P. patens. Phenotypes shown by protonemata and gametophores treated with 1 µM 17-β-estradiol (ER): PpVNS1 overexpressor ER-PpVNS1 (A to D), PpVNS2 overexpressor ER-PpVNS2 (E to H), PpVNS3 overexpressor ER-PpVNS3(I to L), PpVNS4 overexpressor ER-PpVNS4 (M to P), PpVNS5 overexpressor ER-PpVNS5 (Q to T), PpVNS6 overexpressor ER-PpVNS6 (U to X), PpVNS8 overexpressor ER-PpVNS8 (Y to BB), and AtVND7 overexpressor ER-AtVND7 (CC to FF). (B, F, J, N, R, V, and Z) Magnified views of colonies shown in (A, E, I, M, Q, U, and Y), respectively; (D, H, L, P, T, X, and BB) Leaves from gametophores shown in (C, G, K, O, S, W, and AA), respectively. Scale bars, 1 mm (C, G, K, O, S, W, and AA); 500 µm (A, E, I, M, Q, U, and Y); and 100 µm (B, D, F, H, J, L, N, P, R, T, V, X, Z, and BB).

23

Fig. S14. Cell death induced by overexpression of PpVNS7 in protonemata. (A to F) Chloroplast degradation and shrinkage of protoplasm in protonemata: 12 h (A and B) or 24 h (C and D) after incubation with 17-β-estradiol (ER) and mock treatment with DMSO (24 h, E and F). (G to J) Loss of plasma membrane integrity indicated by Evans Blue staining: mock (24 h, G), 0 h (H), 12 h (I), and 24 h (J). Scale bars, 100 µm. Degradation of chloroplasts, shrinkage of protoplasm away from the cell wall, and loss of plasma membrane integrity were prominently observed 12 h after incubation, while some tip cells were living even 24 h after incubation.

24

Fig. S15. Cell death induced by stress treatment in protonemata. Protonemata of PpVNS7 overexpressor were pre-exposed to UV for 30 min (A to D) or treated without (E to H) or with 1 µM 17-β-estradiol (ER) (I to L), and then incubated at

. Evans Blue staining (B, D, F, H, J, and L) indicated all the treatments led loss of plasma membrane integrity after 12 h (B, F, and J), while chloroplast degradation and shrinkage of protoplasm were observed only in the protonemata treated with ER after 24 h (K). Scale bar, 100 µm.

25

Fig. S16. Enriched molecular function GO terms for genes upregulated at least 4-fold in P. patens overexpressing PpVNS7 (2,910 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to ion/metal binding, acyl-CoA oxidase activity, acyl-CoA dehydrogenase activity, and transcription factor activity are significantly enriched (see supplementary table S8for the details).

5.00E-2 < 5.00E-7

hydrolase activity, acting on glycosyl bonds

oxidoreductase activity

carbon-oxygen lyase activity

aconitate hydratase activity

hydrolase activity

hydro-lyase activity

catalytic activity

transferase activity

lyase activity

zinc ion binding

transition metal ion binding

metal ion binding

ion binding

transferase activity, transferring glycosyl groups

xyloglucan:xyloglucosyl transferase activity

transferase activity, transferring hexosyl groups

acyl-CoA dehydrogenase activity

oxidoreductase activity, acting on the CH-CH group of donors

oxidoreductase activity, acting on the CH-CH group of donors,

oxygen as acceptor

acyl-CoA oxidase activity

transcription factor activity

DNA binding

nucleic acid binding

binding

cation binding

ligase activity

ligase activity, forming carbon-sulfur bonds

transcription regulator activity

molecular function

26

Fig. S17. Enriched biological process GO terms for genes upregulated at least 4-fold in P. patens overexpressing PpVNS7 (2,910 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to several catabolic process such as organic acid catabolic process and carboxylic acid catabolic process, as well as several metabolic processes are highly enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

regulation of biological process

cellular amino acid metabolic process oxoacid metabolic process

cellular amine metabolic process

amine metabolic process

monocarboxylic acid metabolic process

fatty acid metabolic process

lipid metabolic process

organic acid metabolic process

cellular catabolic process

cofactor catabolic process

organic acid catabolic process

lipid catabolic process

cellular lipid metabolic process

regulation of nitrogen compound metabolic process

regulation of macromolecule biosynthetic process

regulation of macromolecule metabolic process

regulation of nucleobase, nucleoside, nucleotide and nucleic

acid metabolic process

regulation of primary metabolic process

regulation of metabolic process

regulation of biosynthetic process

regulation of cellular metabolic process

regulation of cellular biosynthetic process

cellular amino acid catabolic process

nitrogen compound metabolic process

cellular nitrogen compound metabolic process

regulation of cellular process

cellular amino acid and derivative metabolic process

amine catabolic process

metabolic process

biological regulationfatty acid catabolic process

fatty acid oxidation

lipid oxidation

fatty acid beta−oxidation

cellular lipid catabolic process

lipid modification

regulation of transcription

response to chemical stimulus

regulation of transcription, DNA−dependent

regulation of RNA metabolic process

carbohydrate metabolic process

regulation of gene expression

cellular process primary metabolic process

response to stimulus

cellular carbohydrate metabolic process

small molecule catabolic process

carboxylic acid metabolic process

carboxylic acid catabolic process

catabolic process

cellular metabolic process

small molecule metabolic process

cofactor metabolic process

cellular ketone metabolic process

alcohol metabolic process

biological process

27

Fig. S18. Enriched cellular component GO terms for genes upregulated at least 4-fold in P. patens overexpressing PpVNS7 (2,910 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms cell wall and external encapsulating structure are highly enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

cell

microbody

organelle

intracellular membrane-bounded organelle

membrane-bounded organelle

peroxisome

cell part

intracellular part

cytoplasm

cell wall

intracellular

intracellular organelle

external encapsulating structure

cytoplasmic part

cellular component

28

Fig. S19. Estrogen-induced overexpression of PpVNS genes can induce xylem vessel-like formation in A. thaliana. Ten-day-old seedlings were treated with 10 µM 17-β-estradiol (ER) for 4 days. (A) Wild-type leaves after 17-β-estradiol treatment. (B to I) Leaves of transgenic lines carrying the constructs for estrogen-inducible PpVNS1 (B), PpVNS2 (C), PpVNS3 (D), PpVNS4 (E), PpVNS5 (F), PpVNS6 (G), PpVNS7 (H), and PpVNS8 (I), respectively. Yellow arrowheads indicate ectopic secondary cell wall deposition. Scale bar, 50 µm.

29

Fig. S20. Estrogen-induced overexpression of PpVNS genes can induce xylem vessel formation-related genes in A. thaliana. qRT-PCR results showing upregulation of MYB46, a transcriptional activator for secondary wall formation (A), in addition to CesA4, a secondary wall-specific CesA gene (B), and XCP1, a papain-like cysteine peptidase gene (C) by 17-β-estradiol treatment after 24 h (n=3). PpVNS7 overexpression upregulated other remaining two secondary wall-specific CesA genes, CesA7 and CesA8 (D), whereas primary wall-specific CesA genes, CesA1, CesA3, and CesA6, were downregulated (E) by PpVNS7 overexpression. Welch’s t-test; * P < 0.05 and ** P < 0.01. Error bars show s.d..

30

Fig. S21. Enriched plant slim GO terms for genes downregulated at least 4-fold in P. patens overexpressing PpVNS7 (3,724 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to broad aspects, especially those related to photosynthesis and chloroplast, are enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

intracellular

thylakoid

plastid

cytoplasm cell

cellular component

photosynthesis

cellular process

response to abiotic stimulus

secondary metabolic process



response to external stimulus

biosynthetic process


metabolic process

biological process

generation of precursor metabolites and energy


motor activitycatalytic activity

hydrolase activity

molecular function

31

Fig. S22. Enriched molecular function GO terms for genes downregulated at least 4-fold in P. patens overexpressing PpVNS7 (3,724 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. A number of GO terms related to catalytic activity including oxidoreductase activity, isomerase activity, and transferase activity are significantly enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

ion transmembrane transporter activity active transmembrane transporter

activity

transmembrane transporter activity

substrate−specific transmembrane transporter activity

substrate−specific transporter activity

transporter activity

fructose 1,6−bisphosphate 1−phosphatase activity

hydrolase activity

ribose phosphate diphosphokinaseactivity

phosphatase activity

phosphoric ester hydrolase activity

hydrolase activity, acting on esterbonds

hydrolase activity, acting oncarbon−nitrogen (but not peptide)

bonds, in cyclic amidines

hydrolase activity, acting on acid anhydrides

diphosphotransferase activity

carbohydrate phosphatase activity

hydrolase activity, acting oncarbon−nitrogen (but not peptide)

bonds

hydrolase activity, acting on acid anhydrides, in

phosphorus−containing anhydrides

motor activity

pyrophosphatase activity

isomerase activity

carbon−carbon lyase activity

glyceraldehyde−3−phosphatedehydrogenase (NADP+)(phosphorylating) activity

glyceraldehyde−3−phosphatedehydrogenase activity

intramolecular oxidoreductaseactivity

carboxy−lyase activity

nucleoside−triphosphatase activity

methyltransferase activity

prenyltransferase activity

transferase activity, transferring acylgroups transferase activity, transferring

one−carbon groups

transferase activity, transferring acylgroups other than amino−acyl

groupsfatty acid synthase activity

geranyltranstransferase activity

acyltransferase activity

binding

RNA binding

ion binding

nucleic acid binding

metal ion binding

magnesium ion binding

cofactor binding

coenzyme bindingcation binding

tRNA binding

carboxylic acid transmembrane transporter activity

bile acid:sodium symporter activity

metal ion transmembrane transporter activity

organic acid transmembranetransporter activity

organic acid:sodium symporter activity

monocarboxylic acid transmembrane transporter activity

bile acid transmembrane transporter activity


ligase activity, forming carbon−nitrogen bonds


lyase activity

ligase activity

transferase activity, transferringphosphorus−containing groups

transferase activity, transferringaldehyde or ketonic groups

carbon−nitrogen ligase activity, withglutamine as amido−N−donor

transferase activity, transferring alkyl or aryl (other than methyl)

groups

catalytic activity

symporter activity

solute:cation symporter activity

solute:sodium symporter activity

inorganic cation transmembrane transporter activity

secondary active transmembrane transporter activity

monovalent inorganic cation transmembrane transporter activity

cation transmembrane transporter activitysodium ion transmembrane

transporter activity

oxidoreductase activity, acting onthe aldehyde or oxo group of

donors, NAD or NADP as acceptor

oxidoreductase activity, acting onthe aldehyde or oxo group of

donors

oxidoreductase activity, acting on CH−OH group of donors

oxidoreductase activity, acting on the CH−OH group of donors, oxygen

as acceptor

oxidoreductase activity, acting onthe CH−CH group of donors, NAD or

NADP as acceptor

oxidoreductase activity, acting on the CH−OH group of donors, NAD or

NADP as acceptor

oxidoreductase activity, acting onpaired donors, with incorporation or

reduction of molecular oxygen

oxidoreductase activity, acting on the CH−CH group of donors

molecular function

32

Fig. S23. Enriched biological process GO terms for genes downregulated at least 4-fold in P. patens overexpressing PpVNS7 (3,724 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. A variety of GO terms of biological process are highly enriched, which prominently include GO terms related to photosynthesis and a number of biosynthetic processes (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

thiamin and derivative biosynthetic process

cellular nitrogen compound biosynthetic process

thiamin biosynthetic process

organic acid biosynthetic process

small molecule biosynthetic processthiamin metabolic process

sulfur compound biosynthetic process

heterocycle metabolic process

nitrogen compound metabolic process

primary metabolic process

aromatic compound biosynthetic process

glucose catabolic process

glucose metabolic process

glycolysis

monosaccharide catabolic process

hexose catabolic process


pigment metabolic process

isoprenoid metabolic process

pigment biosynthetic process

isoprenoid biosynthetic process

lipid biosynthetic process

terpenoid metabolic process

cellular nitrogen compound metabolic process

cellular biosynthetic process

carotenoid metabolic process

tetrapyrrole metabolic process

heterocycle biosynthetic process

porphyrin metabolic process

porphyrin biosynthetic process

chlorophyll biosynthetic process

cofactor biosynthetic process

tetrapyrrole biosynthetic process

alcohol catabolic process


photosynthesis, light harvesting

alcohol metabolic process

cellular carbohydrate catabolic process

monosaccharide metabolic process

carbohydrate catabolic process

tetraterpenoid biosynthetic process


tetraterpenoid metabolic process

terpenoid biosynthetic process

chlorophyll metabolic process

carotenoid biosynthetic process

cofactor metabolic process

vitamin biosynthetic process

water−soluble vitamin biosynthetic process sulfur metabolic process

hexose metabolic process

cellular metabolic process

cellular aromatic compound metabolic process

carbohydrate biosynthetic process

water−soluble vitamin metabolic process

vitamin metabolic process

thiamin and derivative metabolic process

catabolic process

small molecule catabolic process

photosynthesis, dark reaction

generation of precursor metabolites and energy


photosynthesis, light reactionreductive pentose-phosphate cycle

photosynthesis



amine metabolic process

nucleic acid metabolic process

metabolic process

fatty acid biosynthetic process


oxidation reduction

nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

establishment of localization

protein import

protein targeting to chloroplast

protein transport

localization

protein targeting to membrane

cellular protein localization

cellular macromolecule localization

protein targeting

intracellular protein transport

protein import into chloroplast thylakoid membrane

response to blue lightresponse to stimulus

response to light stimulus


response to radiation

cellular localization

cellular amino acid metabolic process

cellular macromolecule metabolic process

ncRNA metabolic process

amine catabolic processcarboxylic acid biosynthetic process

cellular amine metabolic process

carboxylic acid metabolic process

monocarboxylic acid metabolic process

fatty acid metabolic process

RNA metabolic process



oxoacid metabolic process

carbon fixation

organic acid metabolic process

cellular process

cellular ketone metabolic process

organic substance metabolic process

macromolecule metabolic process

organic acid catabolic process

establishment of protein localization

macromolecule localization

protein localization

response to red light

response to red or far red light

response to far red lightcarboxylic acid catabolic process

glycine metabolic process

cellular amino acid catabolic process

glycine catabolic process

serine family amino acid catabolic process

establishment of localization in cell

intracellular transport

transport

snoRNA metabolic process

serine family amino acid metabolic process

biological process

33

Fig. S24. Enriched cellular component GO terms for genes downregulated at least 4-fold in P. patens overexpressing PpVNS7 (3,724 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to photosynthesis and chloroplast are enriched significantly (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

ribosome

intracellular

cell

envelope

cell part

organelle part

organelle membrane

organelle envelope

endosome

membrane-bounded organelle

endosomal part

plastid inner membrane

intracellular organelle part

chloroplast inner membrane

intracellular membrane-bounded organelle

nuclear lumen

nucleoplasm

nucleolar part

non-membrane-bounded organelle

non-membrane-bounded organelle

granular component

nucleolus

Cajal bodynucleoplasm part

organellar ribosome

nuclear part

organelle lumen

intracellular

intracellular organelle

intracellular organelle lumen

nuclear body

nucleus

membrane-enclosed lumen

virion

virion part

extracellular region

organelle

apoplast

photosystem II

photosystem I

macromolecular complex

protein complexthylakoid part

cyclin-dependent protein kinase holoenzyme complex

photosystem

photosystem I reaction center

oxygen evolving complex

intracellular part

plastid

organelle inner membrane

organelle subcompartment

chloroplast membraneplastid ribosome

plastid envelopeplastid membrane

glycine cleavage complex

photosynthetic membrane

membrane part

thylakoid

thylakoid membrane

extrinsic to membranemembrane

ribonucleoprotein complex

endosome membrane

plastid part

plastid thylakoid membrane

thylakoid lumen

plastid thylakoid lumen

external side of endosome membrane

chloroplast thylakoid lumen

cytoplasm

chloroplast thylakoid membrane

cytoplasmic part

plastid thylakoid

chloroplast thylakoid

chloroplast envelope

chloroplast stroma

stromule

plastid stroma

plastoglobule

chloroplast part

chloroplast

cellular component

34

Fig. S25. Enriched molecular function GO terms for direct target genes of VND/NST/SND in A. thaliana (448 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to catalytic activity are enriched, which include cellulose synthase activity and phenylalanine ammonia-lyase activity, reflecting active secondary cell wall formation downstream of VND/NST/SND in A. thaliana (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

heme binding

tetrapyrrole binding

transition metal ion binding

ion binding

binding

structural constituent of cytoskeleton

metal ion binding

cation binding

structural molecule activity

iron ion binding

oxidoreductase activity, acting on CH−OH group of donors

antioxidant activity

oxidoreductase activity, acting on peroxide as acceptor

D−arabinono−1,4−lactone oxidase activity

oxidoreductase activity, acting on the CH−OH group of donors, oxygen

as acceptor


peroxidase activity

ATP citrate synthase activity

transferase activity, transferring acyl groups, acyl groups converted into

alkyl on transfer

transferase activity, transferring acyl groups

glucosyltransferase activity

cellulose synthase activity

transferase activity, transferring glycosyl groups

transferase activity, transferring hexosyl groups

cofactor binding

nucleotide binding

adenyl nucleotide binding

FAD binding

nucleoside binding

purine nucleoside binding

purine nucleotide binding

coenzyme bindingpyrophosphatase activity

nucleoside−triphosphatase activity

hydrolase activity, hydrolyzing O−glycosyl compounds

peptidase activity

xylan 1,4−beta−xylosidase activityhydrolase activity, acting on glycosyl

bonds

peptidase activity, acting on L−amino acid peptides

microtubule motor activity

motor activity

protein serine/threonine kinase activity

transferase activity, transferring phosphorus−containing groups

protein kinase activity

kinase activity

phosphotransferase activity, alcohol group as acceptor

hydrolase activitylyase activity

hydrolase activity, acting on acid anhydrides

carbon−nitrogen lyase activity

ammonia−lyase activity

catalytic activity

hydrolase activity, acting on acid anhydrides, in

phosphorus−containing anhydrides


electron carrier activity

phenylalanine ammonia−lyase activity

molecular function

35

Fig. S26. Enriched biological process GO terms for direct target genes of VND/NST/SND in A. thaliana (448 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented (corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to secondary cell wall formation are prominently enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

xylem development

cell surface receptor linked signaling pathway

multicellular organismal development

enzyme linked receptor protein signaling pathway

anatomical structure development

tissue development

system development

phloem or xylem histogenesis

phospholipid catabolic process

signaling

microtubule−based movementsignaling pathway

developmental process

multicellular organismal process

cell development

cellular lipid catabolic process

establishment of tissue polarity

establishment of planar polarityorgan development

organ morphogenesis

transmembrane receptor protein tyrosine kinase signaling pathway

morphogenesis of an epithelium

anatomical structure morphogenesis

epithelium development

tissue morphogenesis

morphogenesis of a polarized epithelium

cellular aromatic compound metabolic process


microtubule−based process

cellular process

cellular amino acid derivative metabolic process

phenylpropanoid metabolic process



cellular response to stimulus

response to stimulus

cellular cell wall organization or biogenesis

cellular response to abiotic stimulus

cellular response to gravity



organophosphate metabolic process

phospholipid metabolic process

catabolic process

lipid catabolic process

cell death

death

cellular developmental process

response to gravity

programmed cell death

cell differentiation

developmental programmed cell death

glucuronoxylan biosynthetic process

cell wall polysaccharide biosynthetic process

cellular cell wall macromolecule metabolic process

secondary cell wall biogenesis

xylan metabolic processcellular component macromolecule

biosynthetic process xylan biosynthetic process

plant−type cell wall biogenesis

glucuronoxylan metabolic process

cell wall macromolecule metabolic process

protein modification process

cell wall macromolecule biosynthetic process

plant−type cell wall organization or biogenesis

cell wall biogenesis

cellular component biogenesis

carbohydrate biosynthetic process

macromolecule metabolic process






metabolic process

primary metabolic process

glucan metabolic processcellular protein metabolic process

polysaccharide metabolic process

cellular macromolecule biosynthetic process

macromolecule modificationcell wall organization or biogenesis

protein metabolic process macromolecule biosynthetic process

cellular macromolecule metabolic process

cellular biosynthetic processcellular metabolic process

phosphorylation

phosphate metabolic process

cellular glucan metabolic process

phosphorus metabolic process

protein amino acid phosphorylation

polysaccharide biosynthetic process

cellular carbohydrate biosynthetic process

post−translational protein modification cellular polysaccharide metabolic

process

cellulose metabolic process

cellulose biosynthetic process

glucan biosynthetic processcell wall polysaccharide metabolic

process

cellular polysaccharide biosynthetic process

hemicellulose metabolic process

biological process

36

Fig. S27. Enriched cellular component GO terms for direct target genes of VND/NST/SND in A. thaliana (448 genes).Node size reflects the number of genes belonging to the category. Colored nodes represent GO terms that are significantly over-represented(corrected p value <0.05), and the color scale indicates increasingly higher statistical significance. GO terms related to cell wall and cytoskeleton are significantly enriched (see supplementary table S8 for the details).

5.00E-2 < 5.00E-7

organelle part

non−membrane−bounded organelle

intracellular organelle partintracellular

non−membrane−bounded organelle

organelle

intracellular part

microtubule cytoskeleton

protein complex

macromolecular complex

tubulin complex

citrate lyase complex

cytoskeleton intracellular organelle

cytoplasmic part

cytoskeletal part

cell wall

anchored to membrane

membrane

intrinsic to membrane

intracellular

external encapsulating structure

cytoplasm

cell

cell part

membrane part

cellular component

37

Fig. S28. Maximum likelihood tree of MYB proteins in plants.(A) Whole view. (B) Magnified view indicated by (b) in (A). On the scale, 0.2 represents the estimated number of substitutions per site. AtMYB46 was underlined with a red line. At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow); Pp: Physcomitrella patens (purple); Vocar, Volvox carteri; Csubellipsoidea, Coccomyxa subellipsoidea; Mpusilla, Micromonas pusilla; Olucimarinus, Ostreococcus lucimarinus.

Potri.008G089700.1 Potri.008G089200.1

Potri.010G165700.1 Potri.017G086300.1 Os08g33660.1 Os04g38740.1

Os02g36890.1 AT3G01140.1|MYB106 AT5G15310.1|MYB16

Pp1s389 40V6.1 Pp1s45 150V6.1

Pp1s1 78V6.1 Pp1s2 60V6.1

Pp1s2 55V6.1 Pp1s389 41V6.1

Pp1s326 37V6.1 Pp1s106 216V6.1

Pp1s317 55V6.1 Pp1s106 224V6.1

Mpo JPYU-8177 AT5G54230.1|MYB49 Potri.011G125900.1

Potri.001G408700.1 Potri.011G041600.1 AT4G05100.1|MYB74

Potri.004G033100.1 AT4G21440.1|MYB102

Os07g37210.1 Potri.013G148600.1 Potri.019G118200.1

AT4G28110.1|MYB41 Potri.012G140700.1 Potri.015G143500.1 Potri.014G081200.1

Potri.002G157600.1 AT3G61250.1|MYB17 Os02g42870.1

Os04g45060.1 AT1G34670.1|MYB93

Potri.005G164900.1 Potri.005G074500.1

Potri.007G093900.1 Potri.002G096800.1 Os08g37970.1

Os06g11780.1 AT5G10280.1|MYB92

AT5G65230.1|MYB53 Potri.019G050900.1

Potri.001G139900.1 Potri.003G094200.1

AT5G16770.1|MYB9 AT3G02940.1|MYB107

Os02g51799.1 AT4G17785.1|MYB39

Os06g02250.1 Os04g50680.1

AT5G60890.1|MYB34 AT1G18570.1|MYB51 AT5G61420.2|MYB28/PMG1

AT5G07700.1|MYB76 AT5G07690.1|MYB29/PMG2

Os03g27090.1 Potri.019G118900.1

Potri.013G149200.1 Potri.019G018200.1 Potri.008G166700.1

Potri.005G224100.1 Potri.002G038500.1

AT2G31180.1|MYB14 AT1G06180.1|MYB13

AT3G23250.1|MYB15 Os04g43680.1

Os02g41510.1 AT1G79180.1|MYB63

AT1G16490.1|MYB58 AT1G56160.1|MYB72

Os02g46780.1 Os04g50770.1

Os10g33810.1 AT1G74430.1|MYB95

Os09g24800.1 Os07g43580.1

Os08g33940.1 Os11g03440.1 Os12g03150.1 Os11g35390.1


AT1G66230.1|MYB20 AT5G16600.1|MYB43

Os08g33150.1 Os09g23620.1

Os06g14670.1 Os02g49986.1

Potri.001G118800.1 Potri.003G114100.1

Potri.012G127700.1 Potri.015G129100.1

AT4G12350.1|MYB42 AT4G22680.1|MYB85

Os09g36250.1 Potri.008G180800.1

Potri.001G336700.1 AT5G14340.1|MYB40

Pp1s433 19V6.1 Pp1s420 4V6.1

Pp1s78 152V6.1 Potri.012G140500.1

AT4G25560.1|MYB18 AT5G52260.1|MYB19

AT3G48920.1|MYB45 Os02g02370.1

Os04g45020.1 Os02g42850.2


Potri.009G061500.1 Potri.001G267300.1

AT3G08500.1|MYB83 Os12g33070.1

Potri.003G132000.1 AT1G63910.1|MYB103

Potri.001G099800.1 Os08g05520.1

Potri.003G155700.1 Potri.001G075400.1

AT3G12720.1|MYB67 Potri.008G081600.1

Potri.010G174500.1 Os07g31470.1

Potri.001G197000.1 AT3G13890.2|MYB26/MS35

Os01g51260.1 Smo 84195

Potri.014G111200.1 Potri.002G185900.1

AT4G01680.3|MYB55 Potri.005G001600.1

Potri.013G001000.1 Potri.010G004300.1

AT5G26660.1|MYB86 AT1G57560.1|MYB50

AT1G09540.1|MYB61 Os05g04820.1

Os01g18240.1 Os07g44090.3

Os03g25550.1 Os05g46610.1 Os01g50720.1


Os01g07450.1 Smo 437219

Pp1s24 48V6.1 Pp1s189 29V6.1

Pp1s277 14V6.1 Pp1s149 164V6.1

Pp1s211 139V6.1 Potri.017G075000.1

Os03g18480.1 Potri.011G167600.1

Os04g39470.1 AT5G56110.1|MYB80/MS188

Pp1s36 198V6.1 Pp1s65 211V6.1

Pp1s257 83V6.1 Os03g56090.1 Os02g54520.1

Os08g15020.1 Os10g35660.1

Os09g26170.1 Os05g48010.1

Os01g49160.1 Os01g09590.1

Os01g52410.1 Mpo JPYU-8557

Pp1s46 302V6.1 Pp1s284 57V6.1 Pp1s204 13V6.1

Pp1s141 40V6.1 Pp1s295 52V6.1

Mpo JPYU-3085 AT2G47460.1|MYB12/PFG1

AT3G62610.1|MYB11 Potri.010G141000.1

AT5G49330.1|MYB111/PFG3 Potri.002G198100.1

Os01g19970.1 Os03g19120.1

Potri.004G174400.1 Potri.009G134000.1

Os09g36730.1 AT4G38620.1|MYB4

Os08g43550.1 Potri.T011400.1 Potri.004G138000.1 AT2G16720.1|MYB7

AT4G34990.1|MYB32 Os05g35500.1 Os01g65370.1 Potri.013G109300.1 Potri.019G081500.1

AT4G09460.1|MYB6 AT1G35515.1|MYB8/HOS10

AT1G22640.1|MYB3 Os12g07640.1

Potri.015G041100.1 Potri.010G114000.1

Potri.008G128500.1 Potri.004G088100.1

Potri.017G128900.1 Potri.005G112000.1

Smo 109587 Mpo JPYU-5184

Potri.018G049600.1 Potri.018G049200.1

Os03g29614.1 AT5G35550.1|MYB123


Potri.018G127700.1 Potri.006G066400.1

AT5G52600.1|MYB82 Potri.006G221500.1

Potri.018G005300.1 Potri.006G275900.1

AT5G14750.1|MYB66/WER AT5G40330.1|MYB23

AT3G27920.1|MYB0/ATGL1 Potri.003G144200.1 Potri.001G086700.1

Potri.003G144300.1 Potri.014G100800.1


Potri.013G056500.1 Potri.013G056400.1

AT3G13540.1|MYB5 Potri.019G036400.1 Potri.019G036300.1

Os01g50110.1 Smo 98669

Pp1s61 196V6.1 Pp1s371 6V6.1

Pp1s6 416V6.1 Pp1s133 87V6.1

AT3G01530.1|MYB57 Os05g49310.1

Os01g45090.1 Os03g20090.1

Os01g19330.1 Os01g03720.1

Os07g48870.1 Os05g04210.1 Os12g37690.1

Os11g45740.1 Os04g42950.1

Os02g40530.1 Os03g04900.1

Os01g74410.1 Os12g37970.1

Os11g47460.1 Os06g40330.1

AT2G32460.1|MYB101 Os03g38210.1

Os06g46560.1 Os01g59660.1

Os05g41166.1 Smo 81112

Smo 84369 Pp1s66 200V6.1

Mpo JPYU-2625 Os04g46384.1

Pp1s16 281V6.1 Pp1s114 136V6.1

Smo 80553 Pp1s74 264V6.1

Csubellipsoidea|fgenesh1 pg.19 * 53|44671 Os09g01960.1

Os02g09480.1 Smo 77255

Smo 99462 Pp1s10 267V6.1

Mpo JPYU-3364 Pp1s72 143V6.1

Mpo JPYU-8380 Pp1s53 216V6.1

Csubellipsoidea|e gw1.3.530.1|12896 Vocar20005463m

Vocar20002419m Pp1s36 43V6.1 Pp1s120 159V6.1


Csubellipsoidea|gw1.10.406.1|7682 Mpusilla RCC299|e gw2.14.322.1|88104

Olucimarinus|e gwEuk.9.410.1|34891 Smo 100734 Pp1s206 27V6.1

Pp1s103 56V6.1 Pp1s186 70V6.1

Pp1s313 81V6.1 Vocar20009071m


Mpusilla RCC299|gw2.16.261.1|74639 Olucimarinus|gwEuk.10.443.1|8749

95

99

72

63

99

99

97

84 41

93

98

100

30

63

43

99

88

90

54

51

97

42

93

94

92

90

99

19

46

77

75

35

100

10

58

45

42

42

24

90

38

32

29

37

4

62

42

25

99

99

8

72

94

96

97

35

17

88

99

21

65

97

100

64

12

100

82

58

42

37

58

65

47

99

64

11

95

35

98

72

59

48

61

79

22

76

85

60

71

92

73

98

99

64

62

93

98

35

73

41

88

0

57

1

99

66

25

9

44

23

97

16

98

8

84

30

69

89

77

39

52

25

65

86

26

56

74

81

99

92

30

93

13

47

70

39

43

40

43

0

36

64

15

0

83

41

27

20

24

62

46

44

60

96

7

6

63

58

86

62

94

81

98

96

42

63

82

25

77

38

95

35

5

60

79

9

10

18

8

10

26

15

59

97

70

86

11

92

96

63

48

21

77

94

37

93

61

5

40

17

28

45

37

7

5

42

59

46

94

61

27

23 52

6

64

44

31

62

38

26

61

57

36

19

43

11

85

61

13

8

10

37

17

79

19

94

13

12

7

34

56

69

57

2

12

18

54

10

38

42

26

14

19

12

13

48

14

15

41

87

16

14

42

18

19

41

21

46

27

10

42

15

41

19

183

5

3

12

6

10

28

11

2

5

38

0.2

(b)

(c)

(d)

Potri.008G089700.1 Potri.008G089200.1

Potri.010G165700.1 Potri.017G086300.1 Os08g33660.1 Os04g38740.1

Os02g36890.1 AT3G01140.1|MYB106 AT5G15310.1|MYB16

Pp1s389 40V6.1 Pp1s45 150V6.1

Pp1s1 78V6.1 Pp1s2 60V6.1

Pp1s2 55V6.1 Pp1s389 41V6.1

Pp1s326 37V6.1 Pp1s106 216V6.1

Pp1s317 55V6.1 Pp1s106 224V6.1

Mpo JPYU-8177 AT5G54230.1|MYB49 Potri.011G125900.1


Potri.004G033100.1 AT4G21440.1|MYB102


AT4G28110.1|MYB41 Potri.012G140700.1 Potri.015G143500.1 Potri.014G081200.1

Potri.002G157600.1 AT3G61250.1|MYB17 Os02g42870.1

Os04g45060.1 AT1G34670.1|MYB93

Potri.005G164900.1 Potri.005G074500.1

Potri.007G093900.1 Potri.002G096800.1 Os08g37970.1

Os06g11780.1 AT5G10280.1|MYB92

AT5G65230.1|MYB53 Potri.019G050900.1

Potri.001G139900.1 Potri.003G094200.1

AT5G16770.1|MYB9 AT3G02940.1|MYB107

Os02g51799.1 AT4G17785.1|MYB39

Os06g02250.1 Os04g50680.1

AT5G60890.1|MYB34 AT1G18570.1|MYB51 AT5G61420.2|MYB28/PMG1

AT5G07700.1|MYB76 AT5G07690.1|MYB29/PMG2

Os03g27090.1 Potri.019G118900.1


Potri.005G224100.1 Potri.002G038500.1

AT2G31180.1|MYB14 AT1G06180.1|MYB13

AT3G23250.1|MYB15 Os04g43680.1

Os02g41510.1 AT1G79180.1|MYB63

AT1G16490.1|MYB58 AT1G56160.1|MYB72

Os02g46780.1 Os04g50770.1

Os10g33810.1 AT1G74430.1|MYB95

Os09g24800.1 Os07g43580.1

Os08g33940.1 Os11g03440.1 Os12g03150.1 Os11g35390.1

Os03g26130.1

9438

42

72

99

100

37

98

99

84

99

92

30

62

46

62

94

81

98

96

42

63

25

77

18

92

77

94

37

59

46

94

61

27

23 52

6

64

44

31

26

61

57

36

85

61

13

8

10

37

17

94

57

2

12

18

54

10

38

41

18

41

46

27

10

42

15

41

19

183

5

3

12

6

10

11

0.2

BA

38

Fig. S28. (cont.)(C and D) Magnified view indicated by (c and d) in (A). On the scale, 0.2 represents the estimated number of substitutions per site. AtMYB46 was underlined with a red line. At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow); Pp: Physcomitrella patens (purple); Vocar, Volvox carteri; Csubellipsoidea, Coccomyxa subellipsoidea; Mpusilla, Micromonas pusilla; Olucimarinus, Ostreococcus lucimarinus.

AT2G47460.1|MYB12/PFG1 AT3G62610.1|MYB11

Potri.010G141000.1 AT5G49330.1|MYB111/PFG3

Potri.002G198100.1 Os01g19970.1


Os09g36730.1 AT4G38620.1|MYB4

Os08g43550.1 Potri.T011400.1 Potri.004G138000.1 AT2G16720.1|MYB7

AT4G34990.1|MYB32 Os05g35500.1 Os01g65370.1 Potri.013G109300.1 Potri.019G081500.1

AT4G09460.1|MYB6 AT1G35515.1|MYB8/HOS10

AT1G22640.1|MYB3 Os12g07640.1

Potri.015G041100.1 Potri.010G114000.1

Potri.008G128500.1 Potri.004G088100.1

Potri.017G128900.1 Potri.005G112000.1

Smo 109587 Mpo JPYU-5184

Potri.018G049600.1 Potri.018G049200.1

Os03g29614.1 AT5G35550.1|MYB123


Potri.018G127700.1 Potri.006G066400.1

AT5G52600.1|MYB82 Potri.006G221500.1

Potri.018G005300.1 Potri.006G275900.1

AT5G14750.1|MYB66/WER AT5G40330.1|MYB23

AT3G27920.1|MYB0/ATGL1 Potri.003G144200.1 Potri.001G086700.1

Potri.003G144300.1 Potri.014G100800.1


Potri.013G056500.1 Potri.013G056400.1

AT3G13540.1|MYB5 Potri.019G036400.1 Potri.019G036300.1

Os01g50110.1 Smo 98669

Pp1s61 196V6.1 Pp1s371 6V6.1

Pp1s6 416V6.1 Pp1s133 87V6.1

AT3G01530.1|MYB57 Os05g49310.1

Os01g45090.1 Os03g20090.1

Os01g19330.1 Os01g03720.1

Os07g48870.1 Os05g04210.1 Os12g37690.1

Os11g45740.1 Os04g42950.1

Os02g40530.1 Os03g04900.1

Os01g74410.1 Os12g37970.1

Os11g47460.1 Os06g40330.1

AT2G32460.1|MYB101 Os03g38210.1

Os06g46560.1 Os01g59660.1

Os05g41166.1 Smo 81112

Smo 84369 Pp1s66 200V6.1

Mpo JPYU-2625 Os04g46384.1

Pp1s16 281V6.1 Pp1s114 136V6.1

Smo 80553 Pp1s74 264V6.1

Csubellipsoidea|fgenesh1 pg.19 * 53|44671 Os09g01960.1

Os02g09480.1 Smo 77255

Smo 99462 Pp1s10 267V6.1

Mpo JPYU-3364 Pp1s72 143V6.1

Mpo JPYU-8380 Pp1s53 216V6.1


Vocar20002419m Pp1s36 43V6.1 Pp1s120 159V6.1



Olucimarinus|e gwEuk.9.410.1|34891 Smo 100734 Pp1s206 27V6.1

Pp1s103 56V6.1 Pp1s186 70V6.1

Pp1s313 81V6.1 Vocar20009071m


Mpusilla RCC299|gw2.16.261.1|74639 Olucimarinus|gwEuk.10.443.1|8749

95

99

72

63

99

99

97

84 41

93

98

100

30

63

43

99

88

90

54

51

42

93

92

99

46

77

75

35

100

10

58

45

42

24

90

32

37

25

99

99

96

97

35

17

88

21

64

12

58

42

58

65

47

99

35

59

61

79

22

64

62

93

98

16

98

30

89

25

65

86

26

56

74

81

93

13

15

41

96

6

63

38

95

35

5

60

79

26

15

59

86

11

96

63

48

21

61

40

17

45

37

5

11

79

19

13

42

26

14

19

12

13

15

16

14

42

19

21

0.2

DC Potri.004G086300.1 Potri.017G130300.1

AT1G66230.1|MYB20 AT5G16600.1|MYB43

Os08g33150.1 Os09g23620.1

Os06g14670.1 Os02g49986.1

Potri.001G118800.1 Potri.003G114100.1

Potri.012G127700.1 Potri.015G129100.1

AT4G12350.1|MYB42 AT4G22680.1|MYB85

Os09g36250.1 Potri.008G180800.1

Potri.001G336700.1 AT5G14340.1|MYB40

Pp1s433 19V6.1 Pp1s420 4V6.1

Pp1s78 152V6.1 Potri.012G140500.1

AT4G25560.1|MYB18 AT5G52260.1|MYB19

AT3G48920.1|MYB45 Os02g02370.1

Os04g45020.1 Os02g42850.2


Potri.009G061500.1 Potri.001G267300.1

AT3G08500.1|MYB83 Os12g33070.1

Potri.003G132000.1 AT1G63910.1|MYB103

Potri.001G099800.1 Os08g05520.1

Potri.003G155700.1 Potri.001G075400.1

AT3G12720.1|MYB67 Potri.008G081600.1

Potri.010G174500.1 Os07g31470.1

Potri.001G197000.1 AT3G13890.2|MYB26/MS35

Os01g51260.1 Smo 84195

Potri.014G111200.1 Potri.002G185900.1

AT4G01680.3|MYB55 Potri.005G001600.1

Potri.013G001000.1 Potri.010G004300.1

AT5G26660.1|MYB86 AT1G57560.1|MYB50

AT1G09540.1|MYB61 Os05g04820.1

Os01g18240.1 Os07g44090.3

Os03g25550.1 Os05g46610.1 Os01g50720.1


Os01g07450.1 Smo 437219

Pp1s24 48V6.1 Pp1s189 29V6.1

Pp1s277 14V6.1 Pp1s149 164V6.1

Pp1s211 139V6.1 Potri.017G075000.1

Os03g18480.1 Potri.011G167600.1

Os04g39470.1 AT5G56110.1|MYB80/MS188

Pp1s36 198V6.1 Pp1s65 211V6.1

Pp1s257 83V6.1 Os03g56090.1 Os02g54520.1

Os08g15020.1 Os10g35660.1

Os09g26170.1 Os05g48010.1

Os01g49160.1 Os01g09590.1

Os01g52410.1 Mpo JPYU-8557

Pp1s46 302V6.1 Pp1s284 57V6.1 Pp1s204 13V6.1

Pp1s141 40V6.1 Pp1s295 52V6.1

Mpo JPYU-3085

97

90

19

42

29

4

62

8

94

65

97

100

8264

11

95

98

72

48

76

85

60

71

92

73

35

73

41

88

0

57

1

99

66

25

9

44

23

97

8

69

77

39

52

47

70

39

43

40

43

0

36

64

0

8327

20

24

44

60

7

58

86

82

9

10

8

10

97

70

93

5

28

7

42

62

38

19

43

12

7

34

56

69

48

14

87

28

2

5

38

0.2

39

Fig. S29. Maximum likelihood tree of cysteine peptidase proteins in plants.(A) Whole view. (B) Magnified view indicated by (b) in (A). On the scale, 0.2 represents the estimated number of substitutions per site. AtXCP1 was underlined with a red line. At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow); Pp: Physcomitrella patens (purple); Vocar, Volvox carteri; Csubellipsoidea, Coccomyxa subellipsoidea; Mpusilla, Micromonas pusilla; Olucimarinus, Ostreococcus lucimarinus.

Potri.007G047600.1 Potri.005G141600.1 AT4G36880.1|RDL1

Potri.009G098100.1 Potri.001G302100.1

AT3G19390.1|RD21C AT3G19400.1|RDL2

Os04g57490.1 Os04g57440.1

Potri.014G024100.1 AT5G43060.1|RD21B

AT1G47128.1|RD21A Smo 184826


Pp1s49 32V6.1|Phypa 208810 Pp1s285 10V6.1|Phypa 224348


Pp1s369 30V6.1|Phypa 63513 Mpo JPYU-10083

Potri.004G207600.1 Potri.005G256000.1 Potri.002G005700.1 AT1G20850.1|XCP2

AT4G35350.1|XCP1 Os01g73980.1 Os05g01810.1

Os02g48450.1 Csubellipsoidea|e gw1.14.128.1|18161

Vocar20004479m MicpuC2.estExt Genewise1.C 90150|28221

Vocar20004304m Csubellipsoidea|estExt Genewise1.C 20653|21961


AT1G09850.1|XBCP3 Potri.005G232900.1

Os05g43230.1 Smo 98707

AT4G11320.1|RDL5 AT4G11310.1|RDL4 AT4G23520.1|RDL6

Smo 270344 Potri.012G090900.1

Potri.015G087400.1 Potri.015G087500.1 AT5G50260.1|CEP1

AT3G48340.1|CEP2 AT3G48350.1|CEP3

Potri.008G183100.1 Os08g44270.1

Os11g14900.1 Os01g67980.1


Potri.007G075900.1 Potri.007G076000.1 Potri.007G076100.1 Potri.007G075300.1

Potri.005G088600.1 Potri.011G064900.1

Potri.004G056100.1 Potri.004G056300.1

Potri.004G056500.1 Potri.004G055900.1

Os03g54130.1 AT5G45890.1|SAG12

Os04g13140.1 Os12g17540.1

Os04g13090.1 Smo 230713

Mpo JPYU-6597 AT1G06260.1

AT3G43960.1|RDL3 Smo 89437

Smo 104486 Os09g32230.1

Os09g38920.1 Os09g21370.1

Os04g01710.1 Os01g22670.1

Os01g11830.1 Os01g24560.1 Os01g24570.1

Os01g11840.1 Os01g22680.1

Os01g24550.1 Os01g24600.1

Mpo JPYU-218294

99

100

99

69

100

6285

10092

9698

100

7197

100

99

97

97

83

89

90

96

38

84

97

99

93

48

42

97

9

81

63

61

87

88

31

96

91

43

40

36

51

36

86

52

8656

53

45

6

12

1

22

510

29

58

34

53

12

4

3 16

6

5

19

42

13

6

37

39

25

10

0

0

9

3

2

7

0

1

25

6

3

0

7

0

0.2

Potri.007G047600.1 Potri.005G141600.1 AT4G36880.1|RDL1

Potri.009G098100.1 Potri.001G302100.1

AT3G19390.1|RD21C AT3G19400.1|RDL2

Os04g57490.1 Os04g57440.1

Potri.014G024100.1 AT5G43060.1|RD21B

AT1G47128.1|RD21A Smo 184826





Potri.004G207600.1 Potri.005G256000.1 Potri.002G005700.1 AT1G20850.1|XCP2

AT4G35350.1|XCP1 Os01g73980.1 Os05g01810.1

Os02g48450.1 Csubellipsoidea|e gw1.14.128.1|18161

Vocar20004479m MicpuC2.estExt Genewise1.C 90150|28221

Vocar20004304m Csubellipsoidea|estExt Genewise1.C 20653|21961


AT1G09850.1|XBCP3 Potri.005G232900.1

Os05g43230.1 Smo 98707

AT4G11320.1|RDL5 AT4G11310.1|RDL4 AT4G23520.1|RDL6

Smo 270344 Potri.012G090900.1

Potri.015G087400.1 Potri.015G087500.1 AT5G50260.1|CEP1

AT3G48340.1|CEP2 AT3G48350.1|CEP3

Potri.008G183100.1 Os08g44270.1

Os11g14900.1 Os01g67980.1


Potri.007G075900.1 Potri.007G076000.1 Potri.007G076100.1 Potri.007G075300.1

Potri.005G088600.1 Potri.011G064900.1

Potri.004G056100.1 Potri.004G056300.1

Potri.004G056500.1 Potri.004G055900.1

Os03g54130.1 AT5G45890.1|SAG12

Os04g13140.1 Os12g17540.1

Os04g13090.1 Smo 230713

Mpo JPYU-6597 AT1G06260.1

AT3G43960.1|RDL3 Smo 89437

Smo 104486 Os09g32230.1

Os09g38920.1 Os09g21370.1

Os04g01710.1 Os01g22670.1

Os01g11830.1 Os01g24560.1 Os01g24570.1

Os01g11840.1 Os01g22680.1

Os01g24550.1 Os01g24600.1

Mpo JPYU-2182 Os01g42780.1

Os01g42790.1 Os09g39090.1

Os09g39070.1 Os09g39120.1

Os09g39160.1 Os09g39100.1

Os09g39110.1 AT1G29090.1

AT2G34080.1 AT1G29080.1

AT3G49340.1 AT2G27420.1

Os07g01800.1 Os06g38450.1

Smo 268054 Smo 143126 Smo 83176 Smo 83554 Smo 230602

Smo 139792 Smo 71198

Smo 78855 Smo 78186

Smo 24001 Smo 71220 Smo 71627

Smo 75571 AT2G21430.1

AT4G39090.1|RD19A AT4G16190.1 Os04g24600.1 Os02g27030.1

Potri.010G228400.1 Os07g29760.1

AT3G54940.2 Pp1s315 40V6.1|Phypa 198150


Smo 266583 MicpuC2.e gw1.3.456.1|14932 Smo 93661

Smo 110288 AT5G60360.1|AALP/SAG2

AT3G45310.1 Pp1s199 134V6.1|Phypa 143194

Smo 439207 Mpo JPYU-9896 Mpo JPYU-2881

Mpusilla RCC299|estExt Genewise2.C Chr 030652|90296 Mpusilla RCC299|AZW fgenesh2 pg.C Chr 02000228|113461

Csubellipsoidea|estExt Genemark1.C 150018|67334 Csubellipsoidea|estExt fgenesh1 pm.C 40272|52923

Vocar20005152m Csubellipsoidea|estExt Genemark1.C 50167|65409

Pp1s58 52V6.1 Pp1s7 406V6.1

Pp1s79 128V6.1 Mpo JPYU-38053 Csubellipsoidea|estExt Genewise1Plus.C 40555|28006


Pp1s169 91V6.1 Vocar20003561m

Csubellipsoidea|estExt fgenesh1 pg.C 100294|47921 Csubellipsoidea|estExt Genemark1.C 310002|68437100

6581

99

100

99

100

100

63

100

94

77

7095

99

98

99

99

97

100

76

99

69

77

100

83

48

99

82

89

68

58

99

6285

100

66

100

100

57

92

94

96

99

59

100

98

70

99

29

100

7197

100

100

99

50

99

97

76

78

97

83

89

90

50

96

38

84

97

99

93

48

40

42

97

40

92

100

1

9

81

63

61

87

87

63

88

88

31

96

91

43

40

36

51

36

86

52

86

9925

25

56

53

45

6

12

1

22

510

29

58

34

13

53

12

4

3 16

6

5

19

42

13

6

3

6

10

37

39

25

10

0

0

9

10

17

3

2

7

0

1

25

6

3

0

7

8

0

11

0.2

A

(b)

B

(c)

40

Fig. S29. (cont.)(C) Magnified view indicated by (c) in (A). On the scale, 0.2 represents the estimated number of substitutions per site. At, Arabidopsis thaliana (green); Potri, Populus trichocarpa (green); Os, Oryza sativa (blue); Smo, Selaginella moellendorffii (pink); Mpo, Marchantia polymorpha (yellow); Pp: Physcomitrella patens (purple); Vocar, Volvox carteri; Csubellipsoidea, Coccomyxa subellipsoidea; Mpusilla, Micromonas pusilla; Olucimarinus, Ostreococcus lucimarinus.

Os09g32230.1 Os09g38920.1

Os09g21370.1 Os04g01710.1

Os01g22670.1 Os01g11830.1

Os01g24560.1 Os01g24570.1

Os01g11840.1 Os01g22680.1

Os01g24550.1 Os01g24600.1

Mpo JPYU-2182 Os01g42780.1

Os01g42790.1 Os09g39090.1

Os09g39070.1 Os09g39120.1

Os09g39160.1 Os09g39100.1

Os09g39110.1 AT1G29090.1

AT2G34080.1 AT1G29080.1

AT3G49340.1 AT2G27420.1

Os07g01800.1 Os06g38450.1

Smo 268054 Smo 143126 Smo 83176 Smo 83554 Smo 230602

Smo 139792 Smo 71198

Smo 78855 Smo 78186

Smo 24001 Smo 71220 Smo 71627

Smo 75571 AT2G21430.1

AT4G39090.1|RD19A AT4G16190.1 Os04g24600.1 Os02g27030.1

Potri.010G228400.1 Os07g29760.1

AT3G54940.2 Pp1s315 40V6.1|Phypa 198150


Smo 266583 MicpuC2.e gw1.3.456.1|14932 Smo 93661

Smo 110288 AT5G60360.1|AALP/SAG2

AT3G45310.1 Pp1s199 134V6.1|Phypa 143194

Smo 439207 Mpo JPYU-9896 Mpo JPYU-2881

Mpusilla RCC299|estExt Genewise2.C Chr 030652|90296 Mpusilla RCC299|AZW fgenesh2 pg.C Chr 02000228|113461

Csubellipsoidea|estExt Genemark1.C 150018|67334 Csubellipsoidea|estExt fgenesh1 pm.C 40272|52923

Vocar20005152m Csubellipsoidea|estExt Genemark1.C 50167|65409

Pp1s58 52V6.1 Pp1s7 406V6.1

Pp1s79 128V6.1 Mpo JPYU-38053 Csubellipsoidea|estExt Genewise1Plus.C 40555|28006


Pp1s169 91V6.1 Vocar20003561m

Csubellipsoidea|estExt fgenesh1 pg.C 100294|47921 Csubellipsoidea|estExt Genemark1.C 310002|68437100

6581

99

100

99

100

100

63

100

94

77

7095

99

98

99

99

97

76

99

69

77

83

48

99

82

89

68

58

99

6285

66

100

100

57

94

99

59

10070

99

29

100

50

99

76

78

83

50

4040

92

100

1

87

63

88

88

9925

25

34

13

3

6

10

10

17

0

25

7

8

11

0.2

C

41

Fig. S30. Alignment of amino acid sequences of cysteine peptidases. Amino acid sequences of A. thaliana XCP1 and XCP2, and their putative orthologs of P. patens were aligned using ClustalW. Identical residues are highlighted on a black background, and similar residues are shaded in gray. Stars and asterisks indicate the residues of the ERFNIN motif required for the function and the positions of the active site cysteine/histidine residues, respectively.

AT4G35350.1|XCP1 MAFSAPS----LSKFSLLVAISASALLCC---AFARDF-----------SIV---------GYTPEHLTNTD--------AT1G20850.1|XCP2 MALSSPS----RILCFALALSAASLSLSF---ASSHDY-----------SIV---------GYSPEDLESHD--------Pp1s49_32V6.1|Phypa_208810 MGCGGRM--AMVLGLFLVLVLAMG--------WEQGNVGRAD-------AIM---------DYEAHELHSDD--------Pp1s285_10V6.1|Phypa_224348 MGWGRRA-----LGLSLVLLVIAI--------GQQADAGRAN-------AIV---------DYEGNQLHSDD--------Pp1s315_40V6.1|Phypa_198150 MAGRGVL-----LTVLVVFVLAGL-VASL-------------PLRDVIQQVT------------------DGVRVDGSVEPp1s52_60V6.1|Phypa_209158 MESRGLL-----LVGIVVLGFAGF-AASL-------------PTGDTIREVT------------------DDALSNGSVEPp1s199_134V6.1|Phypa_143194 MAPIRHG-----LWVALCVLSAV--AVCQ---GRGLP-----LLEAEIAMVTDLEALASTSAGLFTEILGHS--------Pp1s292_39V6.1|Phypa_224573 MGAVENM--ALVVCLVVALLLCGV--------VANGDV-----------------------IRMPTDVGKDQ--------Pp1s19_362V6.1|Phypa_204314 ---------------------------------------------------------------MTTDLGNER--------Pp1s369_30V6.1|Phypa_63513 MASSTQGGVGAVVVSVAVLLLAGI-ACCYEEDGTSESF-----------------------LHMTTDLEHEN--------

AT4G35350.1|XCP1 -------KLLELFESWMSEHSKAYKSVEEKVHRFEVFRENLMHIDQRNNEINSYWLGLNEFADLTHEEFKGRYL--GLAKAT1G20850.1|XCP2 -------KLIELFENWISNFEKAYETVEEKFLRFEVFKDNLKHIDETNKKGKSYWLGLNEFADLSHEEFKKMYL--GL-KPp1s49_32V6.1|Phypa_208810 -------GMLDVFHQWLERHSRVYHSLSEKQRRFQIFKDNLHYIHNHNKQEKSYWLGLNKFSDLTHDEFRALYL--GI-RPp1s285_10V6.1|Phypa_224348 -------AILDVFHQWLETHSRVYRSLSEKHHRFQIFKENFLYIHAHNKQQKSYWLGLNKFSDLTHQEFRAQYL--GT-KPp1s315_40V6.1|Phypa_198150 QFAHALLGAEKQFESFIKEFGKVYHTVEEYEHRFKVFKSNLLRALKHQALDPTASHGVTMFSDLTEEEFATQYL--GL-KPp1s52_60V6.1|Phypa_209158 QFAHALIGAEKRFESFMKDFGKVYHSVEEYEHRFGVFKSNLLKALKHQALDPTASHGVTMFSDLTEEEFTSKYL--GL-KPp1s199_134V6.1|Phypa_143194 -------RDVLHFAGFAAKYKKEYKTVEELKHRFVTFLESVKLVETHNKGQHSYSLAVNEFADMTFEEFRDSRLMKGE--Pp1s292_39V6.1|Phypa_224573 -------LLAGQFAAWAHKHGKVYSAAEERAHRFLVWKDNLEYIQRHSEKNLSYWLGLTKFADLTNEEFRRQYT--GT-RPp1s19_362V6.1|Phypa_204314 -------LLSEQFGAWAHKHGKVYSSLEEHAHRYMVWKDNLEYIQRHSEKNRSYWLGLTKFADITNDEFRRQYT--GT-RPp1s369_30V6.1|Phypa_63513 -------LLLEQFAAWAHKHGKAYHDAEQCLHRFAVWKDNLAYI-RHSETNRTYSLGLTKFADLTNEEFRRMYT--GT-R

AT4G35350.1|XCP1 PQFSRKRQ----PSANFRY--RDITDLPKSVDWRKKGAVAPVKDQGQCGSCWAFSTVAAVEGINQITTGNLSSLSEQELIAT1G20850.1|XCP2 TDIVRRDEE--RSYAEFAY--RDVEAVPKSVDWRKKGAVAEVKNQGSCGSCWAFSTVAAVEGINKIVTGNLTTLSEQELIPp1s49_32V6.1|Phypa_208810 PAGRAHGL---RNGDRFIY--EDVV-AEEMVDWRKKGAVSDVKDQGSCGSCWAFSAIGSVEGVNAIVTGELISLSEQELVPp1s285_10V6.1|Phypa_224348 PVNRQR------KEANFMY--EDVE-AEPKVDWRLKGAVTDVKDQGACGSCWAFSAVGSVEGVNAIKTGELVSLSEQELVPp1s315_40V6.1|Phypa_198150 RPSALS--------TAPTAEPLPTGDLPPSFDWREKGAVGPVKNQGSCGSCWAFSTTGAVEGAHFLATGKLLSLSEQQLVPp1s52_60V6.1|Phypa_209158 RPSVLS--------SAPQAPPLPTEDLPPNFDWREKGAVGPVKDQGGCGSCWAFSTTGAVEGAHFLNSGKLVSLSEQQLVPp1s199_134V6.1|Phypa_143194 -QNCSATV------GNH---VLTGESLPKTKDWREEGIVSQVKNQASCGSCWTFSTTGALEAAHAQATGKMVLLSEQQLVPp1s292_39V6.1|Phypa_224573 IDRSRRLKKGRNATGSFRYA---NSEAPKSIDWREKGAVTSVKDQGSCGSCWAFSAVGSVEGINAIRTGDAISLSVQELVPp1s19_362V6.1|Phypa_204314 IDRSKRSKRK----TGFRYA---DSEAPESVDWRKKGAVTTVKDQGSCGSCWAFSAIGSVEGINAIRTGEAVSLSEQELVPp1s369_30V6.1|Phypa_63513 IDRSRRAKRR----TGFRYA---DSEAPESVDWRKNGAVTSVKDQGSCGSCWAFSAVGSVEGINAIRNGEAVSLSEQELV

AT4G35350.1|XCP1 DCDTT-------F-NSGCNGGLMDYAFQYIISTGGLHKEDDYPYLMEEGICQEQKE-DVERVTISGYEDVPENDDESLVKAT1G20850.1|XCP2 DCDTT-------Y-NNGCNGGLMDYAFEYIVKNGGLRKEEDYPYSMEEGTCEMQKD-ESETVTINGHQDVPTNDEKSLLKPp1s49_32V6.1|Phypa_208810 DCDRG-------Q-NQGCNGGLMDYAFDFIIKNGGIDTEEDYPYKATDGQCDEARKETSKVVVIDDYQDVPTKSESSLLKPp1s285_10V6.1|Phypa_224348 DCDRK-------Q-NQGCNGGLMDYAFEFIIKNGGIDTEKDYPYKARDGRCDEGRR-NSKVVVIDDYQDVPTQSESALMKPp1s315_40V6.1|Phypa_198150 DCDHQCDPEEAQACDAGCGGGLMTNAYKYVEEAGGLELESDYPYKGRDGKCQFNP--NKVAAKVSNFTNIPIDEDQVAAYPp1s52_60V6.1|Phypa_209158 DCDHQCDREEADACDAGCNGGFMTNAYQYVEAAGGLELESDYPYEGRDGKCKFDS--NKVAVKVSNFTNIPVDEDQVAAYPp1s199_134V6.1|Phypa_143194 DCAGE-------FNNFGCGGGLPSQAFEYIRYNGGIDTEDSYPYNAKDSQCRFHK--NTIGAQVWDVVNITEGAETQLKHPp1s292_39V6.1|Phypa_224573 DCDKK-------Y-NQGCNGGLMDYAFDFVIQNGGIDTEKDYPYQGYDGRCDVNKM-NARVVTIDSYEDVPENDEEALKKPp1s19_362V6.1|Phypa_204314 DCDLE-------Y-NQGCNGGLMDYAFDFILENGGIDTENDYPYKGLDGRCDNNKK-NAHVVTIDGYEDVPENDEEALKKPp1s369_30V6.1|Phypa_63513 DCDLE-------Y-NQGCNGGLMDYAFDFIIQNGGIDTEKDYPYKGFDGRCDNSKK-NAHVVTIDGYEDVPENDEEALKK

AT4G35350.1|XCP1 ALAH-QPVSVAIEASGRDFQFYKGGVFNGK-CG---TDLDHGVAAVGYGS-S------KGSDYVIVKNSWGPRWGEKGFIAT1G20850.1|XCP2 ALAH-QPLSVAIDASGREFQFYSGGVFDGR-CG---VDLDHGVAAVGYGS-S------KGSDYIIVKNSWGPKWGEKGYIPp1s49_32V6.1|Phypa_208810 AVSK-NPVSVAIEAGGRDFQHYQGGVFTGP-CG---TDLDHGVLAVGYGTDD------DGVNYWIVKNSWGPSWGEKGYIPp1s285_10V6.1|Phypa_224348 ALTK-NPVSVAIEAGGRDFQHYQGGVFTGP-CG---SELDHGVLAVGYGTDD------DGVNYWIVKNSWGPGWGEKGYIPp1s315_40V6.1|Phypa_198150 LIKS-GPLAIGINAE--FMQTYVAGVSCPIFCNK--RNLDHGVLLVGYAEHGFAPARLAYKPYWIIKNSWGPMWGDKGYYPp1s52_60V6.1|Phypa_209158 LIKS-GPLAIGINAE--FMQTYIAGVSCPIFCNK--RNLDHGVLLVGYAERGFAPARLAYKPYWIIKNSWGPNWGDNGYYPp1s199_134V6.1|Phypa_143194 AIATMRPVSVAFEVVH-DFRLYNGGVYTSLNCHTGPQTVNHAVLAVGYGEDE------NGVPYWIIKNSWGADWGMNGYFPp1s292_39V6.1|Phypa_224573 AVAG-QPVSVAIEAGGRDFQLYSGGVFTGR-CG---TDLDHGVLAVGYGS-E------KGLDYWIVKNSWGEYWGESGYLPp1s19_362V6.1|Phypa_204314 AVAG-QPVSVAIEAGGRDFQLYSGGVFTGE-CG---TDLDHGVLAVGYGS-E------GSLDYWIVKNSWGEYWGESGYLPp1s369_30V6.1|Phypa_63513 AVAG-QPVSVAIEAGGRDFQLYAGGVFSGE-CG---TDLDHGVLAVGYGT-E------DGVDYWIVKNSWGEYWGESGYL

AT4G35350.1|XCP1 RMKRNTGKP-E---GLCGINKMASYPTKTK--------------------------------------------------AT1G20850.1|XCP2 RLKRNTGKP-E---GLCGINKMASFPTKTK--------------------------------------------------Pp1s49_32V6.1|Phypa_208810 RMERMGSNSTS---GKCGINIEPSFPIKKGAN----PPPAPPSPPTPVKPPSQCDSSHSCPASSTCCCAFNIGKYCLQWGPp1s285_10V6.1|Phypa_224348 RMERFGSDSTD---GKCGINIEASFPIKKGPN----PPPSPPSPPSPIKPPSQCDNSHSCPASSTCCCAFNIGKYCLQWGPp1s315_40V6.1|Phypa_198150 KICRG-----H---GECGLNTMVSAVAANVDV------------------------------------------------Pp1s52_60V6.1|Phypa_209158 KICRG-----H---GECGLNTMVSAVSASVDA------------------------------------------------Pp1s199_134V6.1|Phypa_143194 NMEMG-----K---NMCGVATCASYPVVPEDLKEGV--------------------------------------------Pp1s292_39V6.1|Phypa_224573 RMQRNLKDD-N-GYGLCGINIEPSYAVKTSPN----PPNPGPTPPSPPPPEVICDKWRTCPAENTCCCTFPVGKSCLAWGPp1s19_362V6.1|Phypa_204314 RMQRNIKDS-NHQFGLCGINIEPSYAVKTSPN----PPNPGPTPPSPSPPEVVCDKWRTCPSENTCCCTFPVGKMCLAWGPp1s369_30V6.1|Phypa_63513 RMKRNMKDS-NDGPGLCGINIEPSYAVKTSPN----PPNPGPTPPSPTPPEVICDKWRTCPSENTCCCTFPMGKMCLAWG

AT4G35350.1|XCP1 -------------------------------------------------------------------AT1G20850.1|XCP2 -------------------------------------------------------------------Pp1s49_32V6.1|Phypa_208810 CCPMESATCCEDHYHCCPSDFPVCNLRAGQCVKSKNNPFGVPMLERTRAKFNWPKVSDDSEKGRASFPp1s285_10V6.1|Phypa_224348 CCPMESATCCEDHYHCCPSDFPVCNLRAGQCLKDKRNPFGVPMLERTPAKFNWPKFSFEEEK-KASFPp1s315_40V6.1|Phypa_198150 -------------------------------------------------------------------Pp1s52_60V6.1|Phypa_209158 -------------------------------------------------------------------Pp1s199_134V6.1|Phypa_143194 -------------------------------------------------------------------Pp1s292_39V6.1|Phypa_224573 CCALDSATCCDDHYHCCPHEYPICNLDAGLCLKGSHDKEGVALMKRTLAHFNWAGAFE---------Pp1s19_362V6.1|Phypa_204314 CCSLDSATCCDDHYHCCPHDYPVCNLAAGLCLKGEHDKEGVALMKRTLAHFNWLGNF----------Pp1s369_30V6.1|Phypa_63513 CCSMDSATCCDDHYHCCPHDYPVCNLAAGLCVKGEHDKEGVALMKRTMAHFNWQGNF----------

★ ★ ★ ★ ★ ★

!

!

42

Fig. S31. Correlation between RNA-seq data and qRT-PCR results. (A and B) qRT-PCR showing upregulation of P. patens CesA (A) and cysteine peptidase (B) genes in PpVNS7 overexpressor by 17-β-estradiol treatment after 12 h (n=3). Welch’s t-test; ** P < 0.01. Error bars show s.d.. (C) A scatter plot of relative expression values obtained by RNA-Seq and qRT-PCR. The values of the CesA and cysteine protease genes shown in (A) and (B) were plotted, and a good correlation between two data was found.

43

Table S1. Gene list of PpVNS

Gene name in

this paper

Accession number

Locus name in Physcomitrella

patens v1.6 (http://www.cos

moss.org/)

Gene name in Physcomitrella patens subsp patens v1.1 in

JGI

Gene name in Shen et

al. (2009)

Gene name in Zhu et al. (2012)

PpVNS1 AB898081 Pp1s182_37V6.1 (Phypa_89372)

fgenesh1_pg.scaffold_18200

0024 PpNAC28 PpNAC030


e_gw1.161.49.1 PpNAC08 PpNAC009


e_gw1.201.156.1 PpNAC13 PpNAC013

PpVNS4 AB898084 Pp1s77_42V6.1 (Phypa_48901) gw1.77.154.1 PpNAC36 PpNAC027


estExt_gwp_gw1.C_2230002 PpNAC26 PpNAC023



PpVNS8 AB898088 Pp1s70_104V6.1 (Phypa_128437) e_gw1.70.85.1 PpNAC24

PpNAC25 PpNAC005

44

Table S2. Summary of expression patterns of PpVNS family genes in gametophytes.

Gene name

Central region of new leaf

Midrib of young

leaf Rhizoid Protonema Others

PpVNS1 + + + - -

PpVNS2 + + + + axillary hair, leaf

PpVNS3 - - - - -

PpVNS4 + + + - stem, axillary hair, basal leaf

PpVNS5 + + + + axillary hair, basal leaf

PpVNS6 + + - - -

PpVNS7 + + + - axillary hair

PpVNS8 - + + + margin of new leaf, axillary hair

GUS activity detected: +; undetected: -.

45

Table S3. Wilting rate after lower humidity treatment.

Experiment Plants Number of plants Wilting rate

(%) with wilted leaves

without wilted leaves

1 Wild type 2 34 5.6 1 ppvns1 ppvns6 ppvns7 -8 10 26 27.8 1 ppvns1 ppvns6 ppvns7-35 15 21 41.7 2 Wild type 1 35 2.8 2 ppvns1 ppvns6 ppvns7 -8 13 23 36.1 2 ppvns1 ppvns6 ppvns7-35 17 19 47.2 3 Wild type 5 31 13.9 3 ppvns4-3 22 14 61.1 3 ppvns4-5 23 13 63.9 4 Wild type 7 29 19.4 4 ppvns4-3 16 20 44.4 4 ppvns4-5 18 18 50.0

46

Table S4. Numbers of stereid cells examined for cell wall thickness shown in fig. S10A. Plant Tip Middle Basal Wild type A 11 7 7 Wild type B 14 9 5 ppvns1ppvns6ppvns7-line 8 A 5 6 4 ppvns1ppvns6ppvns7-line 8 B 12 9 5 ppvns1ppvns6ppvns7-line 8 C 7 8 4 ppvns1ppvns6ppvns7-line 35 A 8 12 9 ppvns1ppvns6ppvns7-line 35 B 8 10 7

47

Table S6. Top 40 transcripts with increased expression in P. patens overexpressing PpVNS7.

Gene ID Description Fold

Change (log2)

q-value

Pp1s215_17V6 GDSL-like Lipase/Acylhydrolase superfamily protein 13.3216 0.001554 Pp1s400_32V6 (No putative conserved domains) 13.1404 0.000872 Pp1s141_131V6 Xyloglucan endotransglucosylase hydrolase protein 13.0965 8.45E-05 Pp1s225_84V6 GDSL-like Lipase, Acylhydrolase superfamily protein 13.0206 8.45E-05 Pp1s77_124V6 Caleosin-related family protein 12.8375 8.45E-05 Pp1s6_458V6 PpVNS7 12.7261 8.45E-05 Pp1s298_46V6 Glycosyl hydrolase family 53 12.6800 8.45E-05 Pp1s59_117V6 Aminotransferase class-V 12.6526 8.45E-05 Pp1s115_128V6 BEL1-like homeodomain 12.6373 8.45E-05 Pp1s62_51V6 PLC-like phosphodiesterases superfamily protein 12.6188 0.000641 Pp1s266_66V6 Alpha expansin 12.5857 0.000247 Pp1s27_70V6 Domain of unknown function (DUF1929) / Glyoxal oxidase-related protein 12.4798 0.001554 Pp1s103_109V6 M6 family metalloprotease domain protein 12.4701 8.45E-05 Pp1s336_70V6 C2 domain-containing protein 12.3292 0.000795 Pp1s383_3V6 C2 domain-containing protein 12.2470 0.004888 Pp1s317_21V6 Alpha-L-arabinofuranosidase 12.2367 0.000564 Pp1s401_16V6 PGG domain 12.2361 8.45E-05 Pp1s258_34V6 Pectinesterase / Plant invertase, pectin methylesterase inhibitor-related 12.1733 8.45E-05 Pp1s223_40V6 Phosphoesterase family / Phospholipase C 12.0081 8.45E-05 Pp1s164_55V6 (No putative conserved domains) 11.9346 0.000327 Pp1s136_199V6 Isocitrate lyase 11.7983 8.45E-05 Pp1s60_4V6 (No putative conserved domains) 11.6356 0.000167 Pp1s213_103V6 Transketolase 11.6326 0.006013 Pp1s71_280V6 F-box domain protein 11.4180 8.45E-05 Pp1s199_87V6 Dof domain, zinc finger 11.1034 8.45E-05 Pp1s92_41V6 (No putative conserved domains) 10.9815 8.45E-05 Pp1s152_57V6 Vacuolar iron transporter (VIT) family protein 10.9770 0.000641 Pp1s6_276V6 (No putative conserved domains) 10.9051 8.45E-05 Pp1s31_112V6 Ap2 domain-containing transcription factor 10.8484 0.001998 Pp1s10_401V6 Calcineurin-like phosphoesterase 10.8333 8.45E-05 Pp1s6_175V6 Triose-phosphate transporter family 10.6828 0.000327 Pp1s259_104V6 Ap2 domain-containing transcription factor 10.6248 8.45E-05 Pp1s108_73V6 NPH3 family protein 10.5551 0.002147 Pp1s53_80V6 Ap2 domain-containing transcription factor 10.4814 8.45E-05 Pp1s21_35V6 (No putative conserved domains) 10.4783 0.000948 Pp1s30_337V6 Glycoside hydrolase family 16 10.2163 8.45E-05 Pp1s9_371V6 (No putative conserved domains) 10.1506 8.45E-05 Pp1s26_167V6 Glycosyl hydrolases family 17 10.1411 8.45E-05 Pp1s85_156V6 Domain of unknown function (DUF4378) 10.1221 0.003888 Pp1s102_20V6 l-Allo-threonine aldolase 10.1184 8.45E-05

48

Table S7. Top 40 transcripts with decreased expression in P. patens overexpressing PpVNS7.

Gene ID Description Fold

Change (log2)

q-value

Pp1s99_150V6 Iron permease FTR1 family -9.8669 8.45E-05 Pp1s465_25V6 Iron permease FTR1 family -9.6451 8.45E-05 Pp1s232_57V6 UDP-glucuronosyl and UDP-glucosyl transferase -9.5500 8.45E-05 Pp1s366_9V6 Dienelactone hydrolase family -9.3149 0.007208 Pp1s28_282V6 ABC transporter -9.2895 0.009761 Pp1s491_22V6 Tyrosinase / Polyphenol oxidase / Protein of unknown function (DUF_B2219) -9.2378 0.001703 Pp1s19_140V6 Protein tyrosine kinase / Jacalin-like lectin domain -8.9930 8.45E-05 Pp1s359_22V6 Cyclin D -8.8979 8.45E-05 Pp1s10_396V6 chromosome segregation protein -8.6908 0.002368 Pp1s195_22V6 Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase -8.6796 0.009761 Pp1s22_48V6 Cleavage site for pathogenic type III effector avirulence factor Avr -8.6148 0.008041 Pp1s249_22V6 Iron /ascorbate family oxidoreductase -8.5897 8.45E-05 Pp1s545_14V6 Fasciclin domain -8.5632 8.45E-05 Pp1s70_173V6 Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT) -8.3771 8.45E-05 Pp1s48_70V6 2OG-Fe(II) oxygenase superfamily -8.3592 0.000718 Pp1s67_56V6 Copper transport protein ATOX1-related -8.2531 0.000167 Pp1s281_16V6 3-dehydroquinate synthase -8.0155 0.006576 Pp1s107_188V6 PsaD, photosystem I subunit II -7.9962 8.45E-05 Pp1s251_59V6 Beta-expansin -7.9557 8.45E-05 Pp1s106_138V6 (No putative conserved domains) -7.9250 8.45E-05 Pp1s11_253V6 UDP-D-glucuronate 4-epimerase -7.8693 8.45E-05 Pp1s2_334V6 Cache domain -7.8339 0.005598 Pp1s27_307V6 Mitochondrial carrier protein -7.7777 0.003164 Pp1s108_171V6 Short chain dehydrogenase -7.7490 8.45E-05 Pp1s103_89V6 Serine/threonine protein kinase -7.7201 0.007481 Pp1s20_273V6 Phosphoribulokinase -7.6920 8.45E-05 Pp1s2_226V6 Cold acclimation protein WCOR413 -7.6822 0.006929 Pp1s71_89V6 Chaperone protein dnaJ-related -7.6646 8.45E-05 Pp1s513_5V6 Leucine rich repeat / serine/threonine protein kinase -7.6485 8.45E-05 Pp1s7_198V6 Ribulose bisphosphate carboxylase, small chain -7.6198 8.45E-05 Pp1s59_158V6 Syntaxin -7.6131 0.005944 Pp1s33_259V6 Expansin -7.6122 0.000167 Pp1s287_67V6 Tetraspanin family -7.5906 8.45E-05 Pp1s92_84V6 Pectate lyase -7.5503 8.45E-05 Pp1s136_198V6 Glucose-6-phosphate 1-dehydrogenase -7.5427 8.45E-05 Pp1s300_9V6 Copper transport protein ATOX1-related -7.5272 8.45E-05 Pp1s153_72V6 Fructose-1-6-bisphosphatase -7.4925 8.45E-05 Pp1s223_13V6 Protein of unknown function (DUF1499) -7.4917 8.45E-05 Pp1s214_87V6 Chlorophyll A-B binding protein -7.4808 8.45E-05 Pp1s63_27V6 ABC transporter -7.4551 8.45E-05

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Supplementary Materials forscience.sciencemag.org/content/suppl/2014/03/19/... · . Supplementary Materials for . Contribution of NAC Transcription Factors to Plant Adaptation to