Control of proliferation in the haploid meristem by CLE peptide signaling in Marchantia polymorpha

The homeostasis of meristems in flowering plants is maintained by cell-to-cell communication via CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) peptide hormones. In contrast, cell signals that regulate meristem activity remains elusive in bryophytes that maintain apical meristems in the gametophyte (haploid) body and undergo a gametophyte-dominant life cycle. We here show that MpCLE1 confines the proliferative activity of gametophytic meristem and affects the overall size of gametangiophores (reproductive organs) in Marchantia polymorpha, which is in sharp contrast with the meristem-promoting function of its ortholog TDIF/CLE41/CLE44 in Arabidopsis vascular meristems. Expression analysis suggests that MpCLE1 and its receptor gene MpTDR are expressed in distinct patterns across the apical meristem. These data suggest that local CLE peptide signaling may have had a role in regulating cell proliferation in the shoot meristem in the ancestral land plant and acts in both sporophytic and gametophytic meristems of extant plants.


Introduction
Land plants have evolved unique peptide hormones to control various physiological processes including development and stress responses [1,2]. A notable example is CLE (CLAVATA3/ EMBRYO SURROUNDING REGION-related) family peptides involved in various developmental contexts in flowering plants, such as stem cell maintenance in meristems, vascular development, seed formation and growth control in response to environmental cues [3,4]. The 12-13 amino acid CLE peptides are proteolytically processed from precursor proteins encoded by CLE genes [5][6][7]. CLE peptide hormones undergo post-translational modification such as proline hydroxylation and arabinosylation during maturation [8]. Mature CLE peptides bind to specific membrane receptors that transmit signals to direct cell behavior, thereby manifesting cell-to-cell communication [9,10]. For example, the leucine-rich repeat receptor kinase CLV1 (CLAVATA1) is a receptor for the CLV3 peptide in Arabidopsis, participating in the stem cell homeostasis in the shoot apical meristem [11][12][13]. A phylogenetically related receptor, TDR/PXY (TDIF RECEPTOR/PHLOEM INTERCALATED WITH XYLEM), mediates TDIF (tracheary element differentiation inhibitory factor) peptide signaling essential for stem cell maintenance in the vasculature [14]. CLV3 and TDIF peptides possess characteristic residues for exclusive interaction with their specific receptors [15] and represent two major subclasses of CLE peptide family.
Comparative genomics studies have revealed that the repertoire of developmental regulatory genes is conserved among land plants even though body plans vary among different groups [16][17][18]. All land plants undergo alternation of generations where the both haploid (gametophyte) and diploid (sporophyte) phases develop multicellular bodies and one of the two phases is dominant depending on the plant lineage. Phylogenetically, the monophyletic, diploid-dominant vascular plants either nest within a bryophyte grade, or are sister to a clade of bryophytes that possess haploid-dominant life cycles.
In both types of body plans, meristems function as the source of growth by continually providing new undifferentiated cells. This is achieved by functional zonation of meristems: one or few pluripotent stem cells act as a source of rapidly proliferating cells that often undergo specific division orientations giving rise to differentiating cells [19]. CLE peptides function in shoot, root and vascular meristems of Arabidopsis and other vascular plants, by controlling cell division and differentiation. TDIF, encoded by CLE41/44 in Arabidopsis, is involved in three aspects of vascular cell behavior: inhibition of cell differentiation, enhancement of proliferation and control of cell division orientation [5,20,21]. TDR/PXY encodes a leucine-rich repeat receptor kinase (LRR-RK) of which extracellular LRR domain forms a superhelical structure that binds TDIF at its inner surface [8,[22][23][24]. We have previously reported that the TDIF activity in vascular development is conserved in most vascular plants [25]. However, the biological function of TDIF/CLE, as with any other peptide hormones, is poorly understood in bryophytes. An intriguing question is whether the TDIF/CLE peptides in bryophytes control the meristem activity in the gametophytic body. We here investigated the role for TDIF/CLE peptide in the liverwort Marchantia polymorpha, a model bryophyte species [26,27]. Like many other developmental regulatory genes, the CLE gene family is conserved among land plants [28]. The M. polymorpha genome encodes two CLE genes, MpCLE1 and MpCLE2, belonging to two distinct subclasses (H-type including TDIF and R-type including CLV3, respectively) of the CLE family based on the initial amino acid in the mature peptide hormone motif ( Fig 1A, S1 Fig) [25,29]. In addition, two distinct receptors for the CLE peptides, MpTDR and MpCLV1 are encoded in the M. polymorpha genome (Fig 1B) [29]. In contrast, the moss Physcomitrella patens has only R-type CLE genes and CLV1-type receptors. Thus, M. polymorpha provides a model system for studying TDIF/H-type CLE signaling in bryophyte development.

MpCLE1 encodes a TDIF-type CLE peptide
To test if MpCLE1 is functionally equivalent to AtCLE41, a TDIF-encoding gene of Arabidopsis, we generated gain-of-function alleles in Arabidopsis. The effects of constitutive TDIF expression in Arabidopsis have been previously observed [5,15,20,30]. In the first leaves of 14-day-old seedlings, xylem vessels are formed along the leaf vein in wild type (S2 Fig To elucidate the discrepancy between the phylogenetic and functional relationships of MpCLE1 and AtCLE41, we performed amino-acid swapping between the two peptides, which differ at four residues ( Fig 1C; MpCLE1 and AtCLE41). We synthesized four MpCLE1 peptide variants in which one of the four residues is changed to that of the AtCLE41 peptide ( Fig 1C; MpCLE1-2E/-3V/-5S/-11S, respectively). In addition, hydroxyprolines were incorporated in an MpCLE1 variant (MpCLE1-Hyp) to mimic the natural structure of TDIF ( Fig 1C; O indicates hydroxyproline). Among these MpCLE1-variants, MpCLE1-3V and MpCLE1-5S enhanced stele thickening ( Fig 1D) and suppressed xylem differentiation in the leaf vein ( Fig  1E). These data indicate that MpCLE1 is indeed a TDIF-type CLE gene although a minor amino-acid substitution is required to convert MpCLE1 into a functional peptide in Arabidopsis. Since the MpCLE1-type residues (Fig 1C; N3 and A5) differ from all other known CLEs in land plants, acquisition of these residues in MpCLE1 may have occurred in the liverwort lineage.

MpCLE1 peptide suppresses growth of the M. polymorpha thallus
We next analyzed the biological function of MpCLE1 in M. polymorpha. M. polymorpha is a thalloid liverwort and the body (thallus) grows at the apical notches which are indeterminate apical meristems and bifurcate periodically. For the clonal propagation, disc-shaped small progenies called gemmae are formed in gemmae cups that develop at the dorsal side of the thallus near the apical notch (Fig 2A). When mature gemmae were cultivated for 14 days on solid medium supplemented with MpCLE1 peptide, the overall growth of plants was slightly reduced, and thallus lobes were twisted (S3 Fig). Similar and marginally stronger phenotypes were observed with MpCLE1-Hyp peptide (S3 Fig). Unexpectedly, the effects of TDIF were even stronger than those of MpCLE1 peptides (S3 Fig). Transgenic lines overexpressing either MpCLE1 or AtCLE41 formed small and convoluted thalli (Fig 2A and 2B, S3 Fig). Quantification of the area of thalli showed significant reduction of growth in the MpCLE1 overexpression lines (Fig 2C). In addition, MpCLE1 overexpression lines produced fewer gemmae cups and did not form gametangiophores even 2 months after far-red light induction (S3 Fig).
To verify the minimal functional domain of MpCLE1, we produced overexpression lines with C-terminal deletions (MpCLE1 1-420 and MpCLE1 1-417 ). The former contains the 12 amino-acid CLE peptide motif while the latter lacks the C-terminal asparagine residue of the peptide motif, an essential residue for CLE peptide activities [5,23,24]. As expected, MpCLE1 1-420 overexpression but not MpCLE1 1-417 overexpression exhibited growth defects (S3 Fig), supporting the notion that the 12 amino-acid CLE peptide is the functional domain of MpCLE1.

MpCLE1 reduces proliferative activity in the apical notch
To elucidate the cytological function of MpCLE1, we analyzed apical meristem anatomy in 14-day-old plants grown from gemmae. In wild-type meristems, a single apical cell produces derivatives in four planes-dorsal, ventral and two lateral-and each of these primary derivatives undergoes a stereotypical pattern of divisions producing a 'merophyte' [27,32]. Cell divisions within merophytes produce a pattern of cells in the mature thallus, that when viewed in longitudinal section, appear as rows of cell files emanating from the apical meristem. In longitudinal sections of wild-type plants (Fig 2D), proliferative region, which can be characterized by vertical cell division planes in internal tissues, is approximately 200 μm from the tip of the thallus (bracket in Fig 2E). In contrast, the proliferative region in MpCLE1 overexpression plants was reduced to less than 100 μm from the tip (bracket in Fig 2F), with cell differentiation/expansion occurring at a position closer to the apex, resulting in a distortion of thallus growth. We also examined meristem anatomy in transverse sections (Fig 3A-3C). Consecutive transverse sections of wild-type and MpCLE1 overexpression plants were compared at same positions relative to a basal position "0 μm" at which the two lobes flanking the meristem merged ( Fig 3C). In wild type, small cytoplasmically dense cells are persistently observed in all sections examined (0-180 μm) while MpCLE1 overexpression plants initiate cell expansion at the 40 μm position, and larger cells are observed compared to wild type throughout the sections resulting a thickened dorsi-ventral axis (Fig 3A and 3B). Collectively, these data demonstrate MpCLE1 overexpression reduces the size of the proliferative region in the apical notch. To gain a better understanding of the effects of the loss of MpCLE1, we examined the anatomy of Mpcle1-GT85 plants. In longitudinal sections of Mpcle1-GT85 plants, the proliferative region was expanded (n = 2; 266 and 232 μm in length) compared to 200 μm in wild type (bracket in Fig 4A), although the proliferative region was not as clear as in the wild type due to the less organized cell division plane orientation. In contrast, the timing of cell differentiation in epidermal tissue on the dorsal side is not significantly altered in the mutant since the air chamber development started at similar positions from the apical cells ( Fig 4A). Complementation analysis was performed by introducing a 6.2 kb genetic fragment spanning MpCLE1 into the knock-out line, designated as Mpcle1-GT85 gMpCLE1. Compared to the knock-out mutant, the complementation line had a proliferative region similar in size to that of wild type in both internal (n = 2; 198 and 196 μm) and epidermal tissues, but the orientation of cell division planes in internal tissues is still less organized than in wild type ( Fig 4B). Curled thalli were also observed in the complementation line (S4 Fig). Thus, additional genomic regulatory components might be required to fully complement the phenotype. In consecutive transverse sections, dorsi-ventral thickening of thalli was observed in Mpcle1-GT85 plants compared to the complementation line (Fig 4C and 4D). In the consecutive sections, cell expansion continued until the 320 μm position in Mpcle1-GT85 while it ceased at 200 μm in wild type and the Gametangiophores, specialized reproductive structures, are extensions of the vegetative thalloid body and the proliferative activity of apical meristem. Mpcle1-GT85 plants developed larger antherodiophores (male gametangiophores) than wild type (Fig 5A and 5B). Consistent with the increased proliferation in vegetative thalli, stalks are thicker in Mpcle1-GT85 plants compared to wild type (1.12 ± 0.14 mm v.s. 0.65 ± 0.14 mm, mean ± S.D., n = 10) and are composed of more cells in cross sections (Fig 5C and 5D). The diameter of the antheridial receptacle was also increased in both longitudinal and transverse axes in Mpcle1-GT85 (Fig 5E and  5F, S6 Fig). The mutant receptacles were thicker and contained larger antheridia, which can reach 1.12 mm in length at the maxima (mean ± S.D. = 0.80 ± 0.14 mm, n = 28) in contrast to wild-type antheridia of 0.70 mm in length at the maxima (mean ± S.D. = 0.55 ± 0.08 mm, n = 17) in our observation (Fig 5G-5J), which is consistent to Higo et al. [33]. The complementation line developed normal antheridiophores and antheridia of 0.66 mm in length at the maxima (mean ± S.D. = 0.56 ± 0.08 mm, n = 19) (S4 Fig). Gametangiophore overgrowth was also consistently observed in archegoniophores of female Mpcle1-GT85 plants obtained by cross with Tak-2, without affecting the branching pattern of fingered-lobes (Fig 6H and 6I). These observations support the notion that MpCLE1 negatively controls the proliferative activity in apical meristems.

Genetic interaction of MpCLE1 and MpTDR
To examine the ligand-receptor relationship between TDIF and TDR in M. polymorpha, we analyzed the physiological function of the TDR ortholog, MpTDR, by generating a knock-out line via homologous recombination (S4 Fig; Mptdr-GT400). Similar to Mpcle1-GT85, 21-dayold Mptdr-GT400 thalli were curled upward at the periphery (Fig 6A). In this mutant background, introduction of an MpCLE1 overexpression transgene did not alter thallus morphology ( Fig 6B). The area of thalli was not significantly changed between Mptdr-GT400 and MpCLE1 overexpression in Mptdr-GT400 background (Fig 6C), indicating that MpCLE1 activity is dependent on MpTDR. Consistently, development of Mptdr-GT400 thalli was insensitive to 10 μM TDIF, the most effective MpCLE1-type peptide in our assays (Fig 6D-6G). The archegoniophores of Mptdr-GT400 plants were larger than wild type, resembling the female Mpcle1-GT85 phenotype (Fig 6H-6J). Introduction of MpCLE1 overexpression did not significantly alter the archegoniophore morphology of Mptdr-GT400 (Fig 6K). Collectively, these data indicate that MpCLE1 acts through MpTDR to restrict the proliferative activity in the meristems of M. polymorpha.

Expression of MpCLE1 and MpTDR in apical meristem
To analyze the expression patterns of MpCLE1 and MpTDR, we constructed GUS-reporter lines using 5 kb of genomic sequence upstream of the MpCLE1 and MpTDR coding sequences, respectively. GUS signal for both MpCLE1 and MpTDR promoters was first detected in the apical notches in 5-day-old gemmalings (Fig 7A and 7B). In 10-day-old gemmalings, pro Mp-CLE1:GUS signal was also detected along the midrib in addition to the signals at the apical notches (S7 Fig). Both pro MpCLE1:GUS and pro MpTDR:GUS signals were observed in the developing antheridiophores suggesting that MpCLE1 signaling is functional during the development of antheridiophores (S7 Fig). In longitudinal sections, pro MpCLE1:GUS signal was detected in a small area around the apical cell in 5-day-old gemmalings ( Fig 7C). In contrast, pro MpTDR:GUS signal was detected in the dorsal part in the apical meristem (Fig 7D), suggesting that MpCLE1 signaling may operate within the apical meristem. Since the expression of pro MpTDR:GUS was observed in cells close to the apical cell, the expression domains of MpCLE1 and MpTDR could partially overlap. The influence of MpCLE1 signaling on the expression domains was examined by peptide treatment. In 5-day-old gemmalings grown in the presence of 10 μM MpCLE1-Hyp or TDIF peptide, pro MpCLE1:GUS signals were not conspicuously affected, while pro MpTDR: GUS signals appeared more intense than in the control, however, no change in expression levels was detected in fluorometric quantification assays (S7 Fig). In whole-mount in situ hybridization (WISH) assays, MpCLE1 expression was detected at the apical notches of 7-day-old gemmalings (S8 Fig). MpEF1α-as signal was detected throughout the thallus and strongly in apical notches while MpEF1α-s was not, consistent with Althoff et al. [34] (S8 Fig). Collectively, these data suggest that the MpCLE1 peptide signal may move from a ventral region towards more dorsal regions, with a maximum potential response, i.e. MpTDR expression, near the apical cell.

Discussion
In this work, we show that TDIF-type CLE peptide signaling restricts proliferative activity in the meristem and the overall size of reproductive organs in the liverwort M. polymorpha although development of apical cell, merophyte, or cell division markers such as labile cyclin-GUS is important to unambiguously quantify the change of proliferative activity and its location in the meristem [35]. TDIF has been known as a positive regulator of meristem activity in vascular plants, but conversely MpCLE1/TDIF acts as a negative regulator in M. polymorpha. This function resembles CLV3 peptides in flowering plants. In Arabidopsis clv3 mutants, the shoot and floral meristems are enlarged, resulting in club-shaped siliques and additional floral organs [36]. Tomato mutants and cultivars deficient in CLV3 signaling have excess floral organs and fasciated fruits [37]. In monocots, loss of CLV signaling enhances the meristem activity, which results in the increase of floral organ number in rice and kernel row number in Control of proliferation in the haploid meristem by CLE peptide signaling in Marchantia polymorpha maize [38][39][40][41][42][43]. Since the reproductive structures are determinate in these plants, the effects of enhanced proliferative activities are more conspicuous than effects on vegetative growth. Correlation of proliferative activity in the meristem and reproductive structure size was also found in Mpcle1 mutant that exhibits gametangiophore enlargement along with the enhanced proliferative activity. In contrast to the phenotypes in flowering plants, the gametangiophores of the M. polymorpha mutants did not form an excess number of reproductive structures, such as additional lobes in the receptacles or fasciated stalks. The lobes and stalks are both sexual extensions of thallus and the numbers of these structures depend on the branching of the meristems during gametangiophore development. Thus, the control of proliferative activity by MpCLE1 signaling is uncoupled from meristem branching. In addition to the functional similarity, the expression pattern of MpCLE1 relative to its receptor gene, MpTDR, also resembles to that of CLV3 relative to CLV1 in Arabidopsis, in which the peptide ligand is expressed at/ around stem cells while the receptor is expressed in a neighboring region (Fig 7E) [44,45]. In Arabidopsis, receptor expression in the partially overlapping domain with the ligand is thought to establish different cell fates among neighboring target cells [11,12,46]. In M. polymorpha, proliferative cells in the dorsal region of apical meristem could also interpret MpCLE1 signals from apical cells as a positional cue. However, the morphological phenotypes of knock-out and overexpression mutants for MpCLE1 were found more significantly in the internal tissue. Further identification of downstream signaling components that suppress proliferative activity would clarify the role of MpCLE1-mediated communication in meristem homeostasis.
The slow but continued thallus growth in MpCLE1 overexpression lines indicates that ectopic MpCLE1 does not terminate stem cell activity, and indeed apical cells are maintained in overexpression lines. This contrasts with Arabidopsis CLV3 overexpression, which terminates shoot growth by abolishing stem cells in the meristem [12,13] and with Arabidopsis TDIF mutants that lose vascular stem cells at a certain frequency [47]. Loss of MpCLE1 also resulted in abnormal cell division planes in the meristem, similar to both loss and ectopic expression of TDIF in Arabidopsis [21,22]. Therefore, the cellular function of MpCLE1 signaling appears to be a combination of CLV3 and TDIF signaling observed in Arabidopsis. Likewise, it was recently demonstrated that CLV3 orthologs in P. patens also regulate both cell proliferation and cell division orientation planes in gametophore shoots and that this function is conserved in Arabidopsis [48]. Coupled with our results, these observations hint that perhaps the ancestral functions of both the TDIF and CLV3 pathways may have encompassed regulation of both cell proliferation and cell division orientation planes.
Since TDIF and TDR are genetically conserved in vascular plants, lack of TDIF and TDR in the moss P. patens suggests that TDIF signaling has been lost within the moss lineage. Thus, TDIF signaling is not an indispensable regulatory module for P. patens development despite its significant contribution to M. polymorpha development, perhaps due to the major differences in body plan between mosses and liverworts [49]. In contrast, M. polymorpha retains both TDIF (MpCLE1) and CLV3 (MpCLE2) orthologs suggesting each possesses unique functions in liverworts.
Our work reveals a general association of CLE peptide signaling with both gametophytic and sporophytic meristems in land plants although it's still unclear if CLE is involved in the control of sporophytic meristems in bryophytes. Meristems in both generations evolved in the ancestral land plant and are not present in algae which do not possess CLE peptide signaling. One major difference in the body plans of these organisms is that the land plant body is built from meristems with 3 or more cutting faces, whereas algal bodies are largely constructed from modifications of filamentous growth so that three-dimensional co-ordination of cell behavior is not required. Thus, mechanisms to focus and control meristem growth evolved concomitantly with meristems, with one mechanism being via CLE peptide-based cell-to-cell communication.

Phylogenetic analysis
Phylogenetic analysis was performed as described previously [25]. Sequences for phylogenetic analysis were summarized in S1 Table. The sequences were first aligned with Clustal X and Bayesian phylogenetic analyses were performed on the alignments using MrBayes 3.2.1 [50].

Plant materials and growth conditions
Columbia-0 (Col-0) line of Arabidopsis thaliana and Takaragaike-1 (Tak-1) and BC3-38 lines of M. polymorpha were used as wild type in phenotypic analyses and as the genetic background for transgenic lines. Growth conditions for Arabidopsis and observation methods of vasculature were as described previously [14]. M. polymorpha plants were grown at 22˚C on halfstrength Gamborg B5 medium (pH 5.5, 1.4% agar) under continuous light. For the induction of gametangiophores, far-red light was supplemented. For the observation of antheridia, antheridial receptacles were dissected with forceps under a stereoscopic microscope (Stemi 2000-CS, Zeiss, Jena, Germany). To measure the area of thalli, gemmae were grown on the half-strength Gamborg B5 medium for 14 days and images of plants were analyzed using ImageJ.

Peptide synthesis
Peptides were synthesized by Fmoc chemistry with a peptide synthesizer (CS136XT, CSBio, CA, USA). Analytically pure peptides were obtained by reverse-phase HPLC.

Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from 11-day-old Arabidopsis seedlings with RNeasy plant mini kit (Qiagen, Hilden, Germany). Three independent RNA samples were used for cDNA synthesis with SuperScript III first-strand synthesis system (Thermo Fisher Scientific, MA, USA). Two technical replicates are made for each RNA sample and the average was used as a single data point. Primers used for qPCR are described in S2 Table. The qPCR assay was performed on a LightCycler 96 system (Roche) using KAPA SYBR FAST qPCR kit (KAPA BIOSYSTEMS, MA, USA). Amounts of cDNA input to the qPCR reactions were normalized by the AtTUA4 expression levels. For the comparison of AtCLE41 and MpCLE1 gene expression levels, the expression levels were normalized by performing qPCR using 10 pg of cloning plasmid (pEN-TR-AtCLE41 or pENTR-MpCLE1) as template. Mean values of 3 samples ± S.D. were indicated.

GUS reporter assay
GUS staining was performed according to Ishizaki et al. [51]. Briefly, M. polymorpha gemmalings grown on agar medium were directly submerged in X-Gluc assay solution containing 50mM sodium phosphate buffer (pH 7.2), 1mM potassium-ferrocyanide, 1mM potassium-ferricyanide, 10mM EDTA, 0.01% Triton X-100 and 1mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid. After vacuum infiltration, samples were incubated for 3-12 hours at 37˚C in dark. GUS-stained samples were cleared with ethanol and mounted with clearing solution (chloral hydrate-glycerol-water, 8:1:2) before imaging with light microscope (Axio Imager.A2, Zeiss). For histological analyses, GUS-stained samples were rinsed with water before fixation. At least 2 independent transgenic lines were examined for each experiment and representative images are shown. Fluorometric quantification of GUS activity was performed according to Ishizaki et al. [56] with minor modifications. Three biological replicates were sampled. For each replicate, 5 gemmalings (5-10 mg in total) grown for 5 days on the half-strength Gamborg B5 medium with or without 10 μM peptide were collected in a microtube, frozen with liquid nitrogen and homogenized with micropestle in 100 uL of GUS extraction buffer, containing 50 mM sodium phosphate (pH 7.2), 10 mM 2-mercaptothanol, 1 mM EDTA and 0.01% Triton X-100. Debris was removed by centrifugation at 13,000 rpm for 5 min at 4˚C. Protein concentration was measured using 5 uL of the protein solution using the TaKaRa

Histology
For preparation of plastic sections, plant samples were trimmed with a razor blade and fixed in FAA solution (50% ethanol: 10% formalin: 5% acetic acid in water). Fixed samples were embedded into Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany) and 4 μm sections were prepared with a rotary microtome (RM2235, Leica, Heidelberg, Germany). Sections were stained with 0.02% toluidine blue or with 0.002% Safranin-O solution for GUS-stained samples and then mounted with Entellan New (Merck Millipore, MA, USA).

Whole mount in situ hybridization (WISH)
WISH was performed by modifying a protocol for Arabidopsis seedlings [57]. For the preparation of RNA probes, MpCLE1 and MpEF1α genes were cloned into pCRII vector (Thermo Fisher) by PCR from M. polymorpha cDNA using primers indicated in S2 Table. Digoxygeninlabeled ribo-probes were synthesized using SP6/T7 RNA polymerases (DIG RNA Labeling Kit, Roche, Basel, Switzerland) after digestion with Xho I/BamH I restriction enzymes, respectively. For the preparation of plant samples, 10-day-old gemmalings were fixed in a 1:1 mixture of heptane and fixative (4% paraformaldehyde (PFA), 15% DMSO and 0.1% Tween-20 in water) for 45 min on a rotary shaker at room temperature (RT). Following fixation, tissues were placed in 100% methanol twice for 5 min and 100% ethanol three times for 5 min to remove chlorophyll and incubated for 30 min in a 1:1 mixture of ethanol and Histo-Clear. After treatment of 100% ethanol, tissues were rehydrated in 75% ethanol (v/v in water), 50% ethanol (v/v in phosphate buffered saline; PBS) and 25% ethanol (v/v in PBS) for 10 min each. Tissues were refixed in the fixative for 20 min and rinsed twice for 10 min in PBST (0.1% v/v Tween-20 in PBS) at RT. For permeabilization of cell wall, tissues were treated with 0.1% cellulase (final concentration 100 μg/ml) and 0.2% macerozyme (final concentration 200 μg/ml) for 30 min at RT and with proteinase K (final concentration 125 μg/ml) for 30 min at RT. After stopping the permeabilization with glycine (final concentration 2mg/ml), tissues were refixed in the fixative and rinsed in PBST for 10 min at RT. For labeling, tissues were hybridized with the DIGprobes (150 μg/ml) in the hybridization solution (50% formamide in 5×saline-sodium citrate buffer (SSC) containing 0.1% Tween-20, 0.1 mg/ml heparin and 0.1mg/ml herring sperm DNA) for 16 hours at 55˚C. The probe mixture was denatured prior to use for 10 min at 80˚C. Hybridized tissues were rinsed in 4×SSC three times for 15 min at 55˚C, 0.1×SSC three times for 20 min at 55˚C and maleic acid buffer (MAB) for 5 min at RT. After washing excessive probe, tissues were incubated in 0.1% boheringer blocking reagent in maleic acid buffer (BBR-MAB) for 30 min at RT, the 1:2,000 diluted anti-digoxigenin-AP Fab fragments (Roche) in BBR-MAB for 2 hours at RT and 0.05% (v/v) Tween-20 in MAB three times for 15 min at RT. Bound ribo-probe was detected by overnight staining with nitroblue tetrazolium (Roche) and bromo-chloro-indolyl phosphate (Roche) for overnight at 4˚C. Stained tissues were photographed under a stereoscopic microscope (MZ16F, Leica). WT, HR and RI indicate wild-type, homologous recombination and random insertion genotypes, respectively. In the screening experiments for HR line, many RI lines are generated and primers, c and d, are used to distinguish HR from RI. Genotyping for Mpcle1 and Mptdr gene-targeting mutants (right). Three different primer sets (a-b, c-x, d-y) for each gene were used in genomic PCR to detect wild-type specific (yellow arrows) and mutant-specific (cyan arrows) products. Lanes (wt and mut) are for the PCR products from wild type (Tak-1) and mutant (Mpcle1-GT85 or Mptdr-GT400) DNA samples, respectively. (B) 14-day-old plants grown from gemmae for complementation test.