LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii.

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged.

Introduction

Land plants are characterized by the alternation of haploid (gametophyte) and diploid (sporophyte) phases within their lifecycle, both of which are multicellular (Niklas and Kutschera, 2010; Bowman et al., 2016). In the earliest diverging bryophyte lineages (liverworts, mosses and hornworts) the free-living indeterminate gametophyte predominates the lifecycle, producing gametes that fuse to form the sporophyte. The sporophyte embryo develops on the surface of the gametophyte, ultimately forming a simple determinate spore-producing axis (Kato and Akiyama, 2005; Ligrone et al., 2012). By contrast, angiosperm (flowering plant) sporophytes range from small herbaceous to large arborescent forms, all developing from an indeterminate vegetative shoot apex that ultimately transitions to flowering, and gametophytes are few-celled determinate structures produced within flowers (Schmidt et al., 2015). A series of developmental innovations during the course of land plant evolution thus simplified gametophyte form whilst increasing sporophyte complexity, with a prolonged and plastic phase of vegetative development arising in the sporophyte of all vascular plants (lycophytes, ferns, gymnosperms and angiosperms).

Studies aimed at understanding how gene function evolved to facilitate developmental innovations during land plant evolution have thus far largely relied on comparative analyses between bryophytes and angiosperms, lineages that diverged over 450 million years ago. Such comparisons have revealed examples of both sub- and neo-functionalization following gene duplication, and of co-option of existing gene regulatory networks into new developmental contexts. For example, a single bHLH transcription factor in the moss Physcomitrella patens regulates stomatal differentiation, whereas gene duplications have resulted in three homologs with sub-divided stomatal patterning roles in the angiosperm Arabidopsis thaliana (hereafter ‘Arabidopsis’) (MacAlister and Bergmann, 2011); class III HD-ZIP transcription factors play a conserved role in the regulation of leaf polarity in P. patens and Arabidopsis but gene family members have acquired regulatory activity in meristems of angiosperms (Yip et al., 2016); and the gene regulatory network that produces rhizoids on the gametophytes of both the moss P. patens and the liverwort Marchantia polymorpha has been co-opted to regulate root hair formation in Arabidopsis sporophytes (Menand et al., 2007; Pires et al., 2013; Proust et al., 2016). In many cases, however, interpreting the evolutionary trajectory of gene function by comparing lineages as disparate as bryophytes and angiosperms has proved challenging, particularly when only a single representative gene remains in most extant taxa – as is the case for the LEAFY (LFY) gene family (Himi et al., 2001; Maizel et al., 2005; Sayou et al., 2014).

The LFY transcription factor, which is present across all extant land plant lineages and related streptophyte algae (Sayou et al., 2014), has distinct functional roles in bryophytes and angiosperms. In P. patens, LFY regulates cell divisions during sporophyte development (including the first division of the zygote) (Tanahashi et al., 2005), whereas in angiosperms the major role is to promote the transition from inflorescence to floral meristem identity (Carpenter and Coen, 1990; Schultz, 1991; Weigel et al., 1992; Blázquez et al., 1997; Souer et al., 1998; Molinero-Rosales et al., 1999). Given that LFY proteins from liverworts and all vascular plant lineages tested to date (ferns, gymnosperms and angiosperms) bind a conserved target DNA motif, whereas hornwort and moss homologs bind to different lineage-specific motifs (Sayou et al., 2014), the divergent roles in mosses and angiosperms may have arisen through the activation of distinct networks of downstream targets. This suggestion is supported by the observation that PpLFY cannot complement loss-of-function lfy mutants in Arabidopsis (Maizel et al., 2005). Similar complementation studies indicate progressive functional changes as vascular plant lineages diverged in that the lfy mutant is not complemented by lycophyte LFY proteins (Yang et al., 2017) but is partially and progressively complemented by fern and gymnosperm homologs (Maizel et al., 2005). Because LFY proteins from ferns, gymnosperms and angiosperms recognize the same DNA motif, this progression likely reflects co-option of an ancestral LFY gene regulatory network into different developmental contexts. As such, the role in floral meristem identity in angiosperms would have been co-opted from an unknown ancestral context in non-flowering vascular plants, a context that cannot be predicted from existing bryophyte data.

The role of LFY in non-flowering vascular plant lineages has thus far been hypothesized on the basis of expression patterns in the lycophyte Isoetes sinensis (Yang et al., 2017), several gymnosperm species (Mellerowicz et al., 1998; Mouradov et al., 1998; Shindo et al., 2001; Carlsbecker et al., 2004; Vázquez-Lobo et al., 2007; Carlsbecker et al., 2013) and the fern Ceratopteris richardii (hereafter ‘Ceratopteris’) (Himi et al., 2001), which has been used as a model of fern development for a number of years (Hickok et al., 1995). These studies reported broad expression in vegetative and reproductive sporophyte tissues of I. sinensis and gymnosperms, and in both gametophytes and sporophytes of Ceratopteris. Although gene expression can be indicative of potential roles in each case, the possible evolutionary trajectories and differing ancestral functions proposed for LFY within the vascular plants (Theissen and Melzer, 2007; Moyroud et al., 2010) cannot be resolved without functional validation. Here we present a functional analysis in Ceratopteris that reveals a stem cell maintenance role for at least one of the two LFY homologs in both gametophyte and sporophyte shoots and discuss how that role informs our mechanistic understanding of developmental innovations during land plant evolution.

Results

The CrLFY1 and CrLFY2 genes duplicated recently within the fern lineage

The LFY gene family is present as a single gene copy in most land plant genomes (Sayou et al., 2014). In this regard, the presence of two LFY genes in Ceratopteris (Himi et al., 2001) is atypical. To determine whether this gene duplication is more broadly represented within the ferns and related species (hereafter ‘ferns’), a previous amino acid alignment of LFY orthologs (Sayou et al., 2014) was pruned and supplemented with newly-available fern homologs (see Materials and methods) to create a dataset of 120 sequences,~50% of which were from the fern lineage (Supplementary file 1–3). The phylogenetic topology inferred within the vascular plants using the entire dataset (Figure 1—figure supplement 1) was consistent with previous analyses (Qiu et al., 2006; Wickett et al., 2014). Within the ferns (64 in total), phylogenetic relationships between LFY sequences indicated that the two gene copies identified in Equisetum arvense, Azolla caroliniana and Ceratopteris each resulted from recent independent duplication events (Figure 1). Gel blot analysis confirmed the presence of no more than two LFY genes in the Ceratopteris genome (Figure 1—figure supplement 2). Given that the topology of the tree excludes the possibility of a gene duplication prior to diversification of the ferns, CrLFY1 and CrLFY2 are equally orthologous to the single copy LFY representatives in other fern species.

Figure 1—figure supplement 1.

Phylogenetic relationships between LEAFY sequences reflect established relationships within vascular plant lineages.

Inferred phylogenetic tree from maximum likelihood analysis of 120 LFY sequences sampled from across extant land plant lineages (liverworts, mosses, hornworts, lycophytes, monilophytes i.e. ferns and allies, gymnosperms, angiosperms) including algal (charophyte) sequences as an outgroup. Bootstrap values are given for each node. Sequences belonging to each lineage are denoted by different colours, as shown. The higher-order topology between vascular plant lineages (lycophytes, monilophytes, gymnosperms and angiosperms) is consistent with expected relationships; a gene duplication event resulting in LFY and NEEDLY clades in gymnosperms has been identified previously (Sayou et al., 2014); and relationships between bryophyte lineages are consistent with differences in the LFY DNA binding site preference, where hornworts and mosses each differ from the preferred site shared by liverworts and vascular plants (Sayou et al., 2014).

Figure 1.

CrLFY1 and CrLFY2 arose from a recent gene duplication event.

Inferred phylogenetic tree from maximum likelihood analysis of 64 LFY amino acid sequences (see Supplementary file 1 for accession numbers) sampled from within the fern lineage plus lycophyte sequences as an outgroup. Bootstrap values are given for each node. The tree shown is extracted from a phylogeny with representative sequences from all land plant lineages (Figure 1—figure supplement 1). The Ceratopteris richardii genome contains no more than two copies of LFY (Figure 1—figure supplement 2; indicated by *). Different taxonomic clades within the fern lineage are denoted by different colours, as shown. The divergence between eusporangiate and leptosporangiate ferns is indicated by arrows.

Inferred phylogenetic tree from maximum likelihood analysis of 120 LFY sequences sampled from across extant land plant lineages (liverworts, mosses, hornworts, lycophytes, monilophytes i.e. ferns and allies, gymnosperms, angiosperms) including algal (charophyte) sequences as an outgroup. Bootstrap values are given for each node. Sequences belonging to each lineage are denoted by different colours, as shown. The higher-order topology between vascular plant lineages (lycophytes, monilophytes, gymnosperms and angiosperms) is consistent with expected relationships; a gene duplication event resulting in LFY and NEEDLY clades in gymnosperms has been identified previously (Sayou et al., 2014); and relationships between bryophyte lineages are consistent with differences in the LFY DNA binding site preference, where hornworts and mosses each differ from the preferred site shared by liverworts and vascular plants (Sayou et al., 2014).

(A) Deduced gene structure of CrLFY1 and CrLFY2 loci. All positions marked are given relative to the ATG start codon. Hybridization probes used in DNA gel blot analysis and relevant restriction sites (EcoRI, HindIII) are marked. CrLFY1 probe 1 (868bp) and CrLFY2 probe 1 (851bp) share 79% sequence similarity and hybridize to exons 2 + 3 of each gene (comprising the conserved LFY DNA binding domain). As such, both probes should hybridize to all members of the LFY gene family. CrLFY1 probe 2 (309bp) and CrLFY2 probe 2 (735bp) hybridize to intron 1 of each gene copy and share no significant sequence similarity. As such, each probe is expected to hybridize to the specific gene copy. (B, C) Gel blot analysis of wild-type genomic DNA, digested with EcoRI or HindIII, electrophoresed on an ethidium bromide stained gel (B), blotted to nylon membrane and hybridized against different probes (C) as described in (A). EcoRI digestion was predicted to generate single hybridizing fragments for both CrLFY1 and CrLFY2, each spanning both probes with minimum expected fragment sizes of ~2.0 kb and ~3.1 kb, respectively. HindIII digestion was predicted to generate a single CrLFY1 hybridizing fragment recognized by both probes with a minimum size of ~1.6 kb. HindIII digestion was predicted to generate a CrLFY2 fragment of ~2.5 kb hybridizing to probes 1 and 2, a separate fragment with a minimum size of 559 bp overlapped by 85 bp of probe 1 (and so potentially undetectable) plus an undetectable fragment of 11 bp. The hybridization patterns observed (C) are consistent with these predictions, with the exon probes cross-hybridizing to predicted fragments of both gene copies (but not to any additional gene fragments) and the intron probes primarily hybridizing to the respective specific gene copy.

Figure 1—figure supplement 2.

The Ceratopteris genome contains only two copies of LFY.

(A) Deduced gene structure of CrLFY1 and CrLFY2 loci. All positions marked are given relative to the ATG start codon. Hybridization probes used in DNA gel blot analysis and relevant restriction sites (EcoRI, HindIII) are marked. CrLFY1 probe 1 (868bp) and CrLFY2 probe 1 (851bp) share 79% sequence similarity and hybridize to exons 2 + 3 of each gene (comprising the conserved LFY DNA binding domain). As such, both probes should hybridize to all members of the LFY gene family. CrLFY1 probe 2 (309bp) and CrLFY2 probe 2 (735bp) hybridize to intron 1 of each gene copy and share no significant sequence similarity. As such, each probe is expected to hybridize to the specific gene copy. (B, C) Gel blot analysis of wild-type genomic DNA, digested with EcoRI or HindIII, electrophoresed on an ethidium bromide stained gel (B), blotted to nylon membrane and hybridized against different probes (C) as described in (A). EcoRI digestion was predicted to generate single hybridizing fragments for both CrLFY1 and CrLFY2, each spanning both probes with minimum expected fragment sizes of ~2.0 kb and ~3.1 kb, respectively. HindIII digestion was predicted to generate a single CrLFY1 hybridizing fragment recognized by both probes with a minimum size of ~1.6 kb. HindIII digestion was predicted to generate a CrLFY2 fragment of ~2.5 kb hybridizing to probes 1 and 2, a separate fragment with a minimum size of 559 bp overlapped by 85 bp of probe 1 (and so potentially undetectable) plus an undetectable fragment of 11 bp. The hybridization patterns observed (C) are consistent with these predictions, with the exon probes cross-hybridizing to predicted fragments of both gene copies (but not to any additional gene fragments) and the intron probes primarily hybridizing to the respective specific gene copy.

CrLFY1 and CrLFY2 transcripts accumulate differentially during the Ceratopteris lifecycle

The presence of two LFY genes in the Ceratopteris genome raises the possibility that gene activity was neo- or sub-functionalized following duplication. To test this hypothesis, transcript accumulation patterns of CrLFY1 and CrLFY2 were investigated throughout the Ceratopteris lifecycle (shown as a schematic in Figure 2 for reference).

Figure 2.

The lifecycle of Ceratopteris richardii.

Ceratopteris propagates in the haploid gametophyte phase of its lifecycle (n) through single-celled spores (A) On spore germination (B) a two-dimensional photosynthetic thallus develops into one of two sexes, a default hermaphrodite (C) which produces eggs and sperm (D) or a hormone-induced male that produces sperm only (E). Eggs are retained on the hermaphrodite thallus, and fertilization results in the development of a diploid (2n) embryo on the gametophyte (F), initiating the sporophyte phase of the lifecycle. The sporophyte establishes a vegetative shoot that initiates leaflike lateral organs (fronds) and roots from its apex (G). The first fronds produced are simple but later fronds become increasingly lobed and dissected (H, I). The sporophyte undergoes a reproductive phase-change and subsequent fronds generate haploid spores by meiosis on their undersides (J), enclosed in a morphologically-distinct curled lamina. Mature spores are dispersed to restart the lifecycle.

The developmental stages sampled spanned from imbibed spores prior to germination of the haploid gametophyte (Figure 3A), to differentiated male and hermaphrodite gametophytes (Figure 3B–D), through fertilization and formation of the diploid sporophyte embryo (Figure 3E), to development of the increasingly complex sporophyte body plan (Figure 3F–K). Quantitative real-time PCR (qRT-PCR) analysis detected transcripts of both CrLFY1 and CrLFY2 at all stages after spore germination, but only CrLFY2 transcripts were detected in spores prior to germination (Figure 3L). A two-way ANOVA yielded a highly significant interaction (F(10,22) = 14.21; p<0.0001) between gene copy and developmental stage that had not been reported in earlier studies (Himi et al., 2001), and is indicative of differential gene expression between CrLFY1 and CrLFY2 that is dependent on developmental stage. Of particular note were significant differences between CrLFY1 and CrLFY2 transcript levels during sporophyte development (Supplementary file 4). Whereas CrLFY2 transcript levels were similar across sporophyte samples, CrLFY1 transcript levels were much higher in samples that contained the shoot apex (Figure 3F,G) than in those that contained just fronds (Figure 3H–K). These data suggest that CrLFY1 and CrLFY2 genes may play divergent roles during sporophyte development, with CrLFY1 acting primarily in the shoot apex and CrLFY2 acting more generally.

Figure 3.

CrLFY1 and CrLFY2 are differentially expressed during the Ceratopteris lifecycle.

(A-K) Representative images of the developmental stages sampled for expression analysis in (L). Imbibed spores (A); populations of developing gametophytes harvested at 5 (B, C) and 8 (D) days after spore-sowing (DPS), comprising only males (B) or a mixture of hermaphrodites (h) and males (m) (C, D); fertilized gametophyte subtending a developing sporophyte embryo (em) (E); whole sporophyte shoots comprising the shoot apex with 3 (F) or five expanded entire fronds attached (G); individual vegetative fronds demonstrating a heteroblastic progression in which frond complexity increases through successive iterations of lateral outgrowths (pinnae) (H–J); complex fertile frond with sporangia on the underside of individual pinnae (K). Scale bars = 100 um (A–E), 5 mm (F–H), 20 mm (I–K). (L) Relative expression levels of CrLFY1 and CrLFY2 (normalized against the housekeeping genes CrACTIN1 and CrTBP) at different stages of development. n = 3; Error bars = standard error of the mean (SEM). Pairwise statistical comparisons (ANOVA followed by Tukey’s multiple comparisons test– Supplementary file 4) found no significant difference in CrLFY2 transcript levels between any gametophyte or sporophyte tissues sampled after spore germination (p>0.05) and no significant difference between CrLFY1 and CrLFY2 transcript levels during early gametophyte development (p>0.05) (B, C). Differences between CrLFY1 and CrLFY2 transcript levels were significant in gametophytes at 8 DPS (p<0.05) (D). CrLFY1 transcript levels were significantly higher in whole young sporophytes (F) and vegetative shoots (G) compared to isolated fronds (H–K) (p<0.05). CrLFY1 transcript levels in whole sporophytes and shoots were greater than CrLFY2, whereas in isolated fronds CrLFY1 transcript levels were consistently lower than CrLFY2 (p<0.05). Asterisks denote significant difference (*, p<0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001) between CrLFY1 and CrLFY2 transcript levels (Sidak’s multiple comparisons test) within a developmental stage. Letters denote significant difference (p<0.05) between developmental stages for CrLFY1 or CrLFY2 (Tukey’s test). Groups marked with the same letter are not significantly different from each other (p>0.05). Statistical comparisons between developmental stages were considered separately for CrLFY1 and CrLFY2. The use of different letters between CrLFY1 and CrLFY2 does not indicate a significant difference.

Spatial expression patterns of CrLFY1 are consistent with a retained ancestral role to facilitate cell divisions during embryogenesis

Functional characterization in P. patens previously demonstrated that PpLFY promotes cell divisions during early sporophyte development (Tanahashi et al., 2005). To determine whether the spatial domains of CrLFY1 expression are consistent with a similar role in Ceratopteris embryo (early sporophyte) development, transgenic lines were generated that expressed the reporter gene B-glucuronidase (GUS) driven by a 3.9 kb fragment of the CrLFY1 promoter (CrLFY1). This promoter fragment comprised genomic sequence encoding the entire published 5’UTR (Himi et al., 2001) plus a further 1910 bp upstream of the predicted transcription start site (Figure 1—figure supplement 2A). In the absence of a genome sequence, repeated attempts to isolate an analogous fragment of CrLFY2 sequence were unsuccessful (see Materials and methods for details). Construct maps plus DNA blot and PCR validation of transgenic lines are shown in Figure 4—figure supplements 1–4. GUS activity was monitored in individuals from three independent transgenic lines, sampling both before and up to six days after fertilization (Figure 4A–O), using wild-type individuals as negative controls (Figure 4P–T) and individuals from a transgenic line expressing GUS driven by the constitutive 35S promoter (35S) as positive controls (Figure 4U–Y). Notably, no GUS activity was detected in unfertilized archegonia of CrLFY1 gametophytes (Figure 4A,F,K) but by two days after fertilization (DAF) GUS activity was detected in most cells of the early sporophyte embryo (Figure 4B,G,L). At 4 DAF, activity was similarly detected in all visible embryo cells, including the embryonic frond, but not in the surrounding gametophytic tissue (the calyptra) (Figure 4C,H,M). This embryo-wide pattern of GUS activity became restricted in the final stages of development such that by the end of embryogenesis (6 DAF) GUS activity was predominantly localized in the newly-initiated shoot apex (Figure 4D,E,I,J,N,O). Collectively, the GUS activity profiles indicate that CrLFY1 expression is induced following formation of the zygote, sustained in cells of the embryo that are actively dividing, and then restricted to the shoot apex at embryo maturity. This profile is consistent with the suggestion that CrLFY1 has retained the LFY role first identified in P. patens (Tanahashi et al., 2005), namely to promote the development of a multicellular sporophyte, in part by facilitating the first cell division of the zygote.

Figure 4—figure supplement 1.

Schematic of CrLFY1pro::GUS and 35S constructs.

Hybridization probes for the GUS and hygromycin resistance (HygR) genes, plus the position of the SpeI and HindIII restriction sites used for DNA gel blot analysis (Figure 4—figure supplement 2) are indicated. Restriction sites used in plasmid construction (see Materials and methods) are also indicated. All nucleotide positions are given relative to the start of the T-DNA right border (RB). Primer sequences are listed in the Key Resources Table.

Figure 4—figure supplement 4.

PCR analysis of 35S positive control line identified a full-length 35S insertion.

(A) Schematic of the 35S construct marking binding sites of genotyping primers used in (B). All positions shown are relative to the GUS ATG. Primer sequences are listed in the Key Resoucres Table. (B) PCR was performed on genomic DNA extracted from three T1 individuals, including the two investigated by gel blot analysis (Figure 4—figure supplement 2). T-DNA specific reactions were performed to amplify the GUS CDS (left) previously identified by gel blot analysis (Figure 4—figure supplement 2) using the same primers as the gel-blot probe, and a PCR product spanning almost the full length of the 35S::GUS::OCS construct (right). Black arrowheads mark the expected size of the target PCR product in each reaction. PCR results indicate the presence of a full length 35S T-DNA in this line.

Figure 4.

The CrLFY1 promoter drives reporter gene expression in proliferating tissues of the developing Ceratopteris embryo.

(A–Y) GUS activity detected as blue staining in developing embryos of three independent CrLFY1 transgenic reporter lines (A–O), a representative negative wild-type control line (P–T) and a representative positive 35S control line (U–Y). Tissues are shown prior to fertilization (A, F, K, P, U), or 2 (B, G, L, Q, V), 4 (C, H, M, R, W), and 6 (D, I, N, S, X) days after fertilization (DAF). In CrLFY1 lines, GUS activity first became visible within the first few divisions of embryo development (but not in surrounding gametophyte tissues) at 2 DAF (B, G, L) and was expressed in cells of the embryo frond as it proliferated (C, H, M). GUS activity was visible in the shoot apex and in frond vascular tissue at 6 DAF (D, I, N), with staining in the shoot apical cell (sac), subtending shoot apex tissues and newly-initiated fronds, including the frond apical cell (fac) (E, J, O). No GUS activity was detected in wild-type samples (P–T), whereas the majority of cells in the constitutively expressing 35S samples stained blue (U–Y). Embryos develop on the surface of the gametophyte thallus when an egg cell (ec) within the archegonium (which comprises a venter (v) and neck cells (nc) to allow sperm entry) are fertilized. After fertilization, the venter forms a jacket of haploid cells known as the calyptra (c) that surrounds the diploid embryo (em). Cell fates in the embryo (embryo frond (fr), embryo foot (ft), root apex (ra) and shoot apex (sa)) are established at the eight-celled stage (Johnson and Renzaglia, 2008), which is around 2 DAF under our growth conditions. Embryogenesis is complete at 6 DAF, after which fronds arise from the shoot apex. Scale bars = 50 μm.

Hybridization probes for the GUS and hygromycin resistance (HygR) genes, plus the position of the SpeI and HindIII restriction sites used for DNA gel blot analysis (Figure 4—figure supplement 2) are indicated. Restriction sites used in plasmid construction (see Materials and methods) are also indicated. All nucleotide positions are given relative to the start of the T-DNA right border (RB). Primer sequences are listed in the Key Resources Table.

CrLFY1 lines AE2, AF3, and AG18 were regenerated from three separate bombardments and so are necessarily independent of one another. Genomic DNA was extracted from four T1 sporophytes (arising from the free fertilization of T1 gametophytes) within each line, digested with SpeI and separated on an electrophoresis gel. Genomic DNA from two T1 sporophytes of 35S line two was digested with HindIII. Gel blot analysis of both constructs was performed with the same two probes, hybridizing to the GUS CDS and hygromycin resistance (HygR) CDS, respectively (Figure 4—figure supplement 1). For each full-length CrLFY1 T-DNA insertion present in the genome a single insert is predicted to result in unlinked hybridization fragments with minimum sizes of ~2.5 kb (HygR) and ~6 kb (GUS). For 35S, a single fragment with a minimum size of ~5.1 kb is predicted per insertion event for both probes (see Figure 4—figure supplement 1). Gel blot results indicate the presence of one full-length T-DNA insertion in 35Spro::GUS line 2. Based on the hybridization fragments obtained, CrLFY1 line AE2 contains two potentially full-length insertions of the CrLFY1pro::GUS cassette and 1–2 insertions of partial fragments, AF3 contains a single potential full length insertion and AG18 contains two potential full length insertions.

(A) Schematic of the CrLFY1 construct marking binding sites of PCR primers used in (B). All positions are given relative to the GUS ATG. Primer sequences are listed in the Key Resources Table. (B) PCR was performed on genomic DNA from the same four individual T1 sporophytes within each line investigated by gel blot analysis (see Figure 4—figure supplement 2). PCR reactions were performed to amplify the native 3.9 kb CrLFY1 promoter as a positive control (row 1) and to amplify T-DNA specific products (rows 2–4) containing the GUS CDS and different lengths of contiguous CrLFY1 promoter sequence (see A). Black arrowheads mark the expected size of the target PCR product in each reaction. PCR analysis identified at least one full-length GUS CDS with ~3.8 kb of CrLFY1 promoter in line AF3 (row 2). Faint PCR products indicate that line AG18 and AE2 probably contain a full-length GUS CDS plus ~3 kb of CrLFY1 promoter sequence (row 3). All lines carry a GUS CDS plus a minimum CrLFY1 promoter length of 875 bp (row 4).

(A) Schematic of the 35S construct marking binding sites of genotyping primers used in (B). All positions shown are relative to the GUS ATG. Primer sequences are listed in the Key Resoucres Table. (B) PCR was performed on genomic DNA extracted from three T1 individuals, including the two investigated by gel blot analysis (Figure 4—figure supplement 2). T-DNA specific reactions were performed to amplify the GUS CDS (left) previously identified by gel blot analysis (Figure 4—figure supplement 2) using the same primers as the gel-blot probe, and a PCR product spanning almost the full length of the 35S::GUS::OCS construct (right). Black arrowheads mark the expected size of the target PCR product in each reaction. PCR results indicate the presence of a full length 35S T-DNA in this line.

Figure 4—figure supplement 2.

DNA gel blot analysis of CrLFY1 and 35S transgenic lines.

CrLFY1 lines AE2, AF3, and AG18 were regenerated from three separate bombardments and so are necessarily independent of one another. Genomic DNA was extracted from four T1 sporophytes (arising from the free fertilization of T1 gametophytes) within each line, digested with SpeI and separated on an electrophoresis gel. Genomic DNA from two T1 sporophytes of 35S line two was digested with HindIII. Gel blot analysis of both constructs was performed with the same two probes, hybridizing to the GUS CDS and hygromycin resistance (HygR) CDS, respectively (Figure 4—figure supplement 1). For each full-length CrLFY1 T-DNA insertion present in the genome a single insert is predicted to result in unlinked hybridization fragments with minimum sizes of ~2.5 kb (HygR) and ~6 kb (GUS). For 35S, a single fragment with a minimum size of ~5.1 kb is predicted per insertion event for both probes (see Figure 4—figure supplement 1). Gel blot results indicate the presence of one full-length T-DNA insertion in 35Spro::GUS line 2. Based on the hybridization fragments obtained, CrLFY1 line AE2 contains two potentially full-length insertions of the CrLFY1pro::GUS cassette and 1–2 insertions of partial fragments, AF3 contains a single potential full length insertion and AG18 contains two potential full length insertions.

CrLFY1 is expressed in dividing tissues throughout shoot development

Both mosses and ferns form embryos, but moss sporophyte development is determinate post-embryogenesis (Kato and Akiyama, 2005; Kofuji and Hasebe, 2014) whereas fern sporophytes are elaborated post-embryonically from indeterminate shoot apices (Bierhorst, 1977; White and Turner, 1995). The Ceratopteris shoot apex comprises a single apical cell that generates daughter cells through asymmetric divisions, and individual lateral organs (fronds and root) arise from their own apical cells specified within the grouped descendants of these daughter cells (Hou and Hill, 2002; Hou and Hill, 2004). CrLFY1 expression in the shoot apex at the end of embryogenesis (Figure 4E,J,O) and elevated transcript levels in shoot apex-containing sporophyte tissues (Figure 3L) suggested an additional role for CrLFY1 relative to that seen in mosses, namely to promote proliferation in the indeterminate shoot apex. To monitor CrLFY1 expression patterns in post-embryonic sporophytes, GUS activity was assessed in CrLFY1 lines at two stages of vegetative development (Figure 5A–O) and after the transition to reproductive frond formation (Figure 5—figure supplement 1A–L). Wild-type individuals were used as negative controls (Figure 5P–T; Figure 5—figure supplement 1M–P) and 35S individuals as positive controls (Figure 5U–Y; Figure 5—figure supplement 1Q–T). In young sporophytes (20 DAF), GUS activity was primarily localized in shoot apical tissues and newly-emerging frond primordia (Figure 5A,F,K), with very little activity detected in the expanded simple fronds produced at this age (Figure 5B,G,L). In older vegetative sporophytes (60 DAF), which develop complex dissected fronds (Figure 5C,H,M), GUS activity was similarly localized in the shoot apex and young frond primordia in two out of the three fully characterized lines (Figure 5D,I,N) and in a total of 8 out of 11 lines screened (from seven independent rounds of plant transformation). GUS activity was also detected in developing fronds in regions where the lamina was dividing to generate pinnae and pinnules (Figure 5E,J,O). In some individuals GUS activity could be detected in frond tissues almost until maturity (Figure 5C). Notably, patterns of CrLFY1 expression were the same in the apex and complex fronds of shoots before (60 DAF) (Figure 5C–E,H–J,M–O) and after (~115 DAF) the reproductive transition (Figure 5—figure supplement 1A–L). Consistent with a general role for CrLFY1 in promoting cell proliferation in the shoot, GUS activity was also detected in shoot apices that initiate de novo at the lamina margin between pinnae (Figure 5Z–AD). Together these data support the hypothesis that LFY function was recruited to regulate cell division processes in the shoot when sporophytes evolved from determinate to indeterminate structures.

Figure 5.

The CrLFY1 promoter drives reporter gene expression in proliferating shoot tissues of the Ceratopteris sporophyte.

(A–Y) GUS activity detected as blue staining in post-embryonic sporophytes from three independent CrLFY1 transgenic reporter lines (A–O), negative wild-type controls (P–T) and positive 35S controls (U–Y). Sporophytes were examined at 20 DAF (A, B, F, G, K, L, P, Q, U, V) and 60 DAF (C–E, H–J, M–O, R–T, W–Y). GUS staining patterns are shown for whole sporophytes (A, C, F, H, K, M, P, R, U, W), shoot apices (arrowheads) (B, D, G, I, L, N, Q, S, V, X) and developing fronds (E, J, O, T, Y). In CrLFY1 sporophytes at 20 DAF (producing simple, spade-like fronds) GUS activity was restricted to the shoot apex (A, F, K) and newly-initiated frond primordia, with very low activity in expanded fronds (B, G, L). In CrLFY1 sporophytes at 60 DAF (producing complex, highly dissected fronds) GUS activity was similarly seen in the apex (C, H, M), but persisted for longer during frond development. Activity was initially detected throughout the frond primordium (D, I, N), before becoming restricted to actively proliferating areas of the lamina (E, J, O). Scale bars = 2 mm (A, F, K, P, U), 500 μm (B, D, G, I, L, N, Q, S, V, X) 10 mm (C, H, M, R, W), 1 mm (E, J, O, T, Y). *=GUS staining in maturing frond. GUS staining patterns were the same in leaves formed after the reproductive transition (Figure 5—figure supplement 1). (Z-AD) Fronds can initiate de novo shoots (white arrowheads) from marginal tissue between existing frond pinnae (Z, AA). GUS activity was detected in emerging de novo shoot apices on CrLFY1 fronds (AB–AD). Scale bars = 10 mm (Z, AA), 500 μm (AB–AD).

GUS activity detected as blue staining in sporophytes producing fronds with spore-bearing morphology (narrowing and elongation of pinnae) from three independent CrLFY1 transgenic reporter lines (A–L); 110–124 DAF), negative wild-type controls (M–P); 113 DAF) and positive 35S controls (Q–T); 110 DAF). Staining patterns were consistent between the three independent CrLFY1 transgenic lines (A, E, I), and were similar to those seen at 60 DAF (Figure 4C, (H,M). GUS activity was observed throughout the shoot apex (B, F, J) and in recently-emerged frond primordia (C, G, K). Activity persisted later in frond development, becoming restricted to developing pinnae (D, H, L). GUS staining was lost from fronds prior to maturity (A, E, I). No endogenous GUS activity was detected in wild-type controls (M–P) whereas activity was detected throughout all non-senescent tissues in the 35S line (Q–T).

Figure 5—figure supplement 1.

CrLFY1 expression patterns are similar in Ceratopteris shoots before and after reproductive phase change.

GUS activity detected as blue staining in sporophytes producing fronds with spore-bearing morphology (narrowing and elongation of pinnae) from three independent CrLFY1 transgenic reporter lines (A–L); 110–124 DAF), negative wild-type controls (M–P); 113 DAF) and positive 35S controls (Q–T); 110 DAF). Staining patterns were consistent between the three independent CrLFY1 transgenic lines (A, E, I), and were similar to those seen at 60 DAF (Figure 4C, (H,M). GUS activity was observed throughout the shoot apex (B, F, J) and in recently-emerged frond primordia (C, G, K). Activity persisted later in frond development, becoming restricted to developing pinnae (D, H, L). GUS staining was lost from fronds prior to maturity (A, E, I). No endogenous GUS activity was detected in wild-type controls (M–P) whereas activity was detected throughout all non-senescent tissues in the 35S line (Q–T).

CrLFY1 regulates activity of the sporophyte shoot apex

To test the functional significance of CrLFY expression patterns, transgenic RNAi lines were generated in which one of four RNAi constructs targeted to CrLFY1, CrLFY2 or both were expressed from the maize ubiquitin promoter (ZmUbi). Construct maps plus DNA blot and PCR validation of transgenic lines are shown in Figure 6—figure supplements 1–5. Genotypic screening identified 10 lines which contained the complete transgene cassette and three lines that contained a fragment of the transgene which included the antibiotic resistance marker but not the RNAi hairpin (Table 1).

Figure 6—figure supplement 1.

Positions of CrLFY RNAi target sequences.

Schematic of CrLFY1 and CrLFY2 transcripts, showing recognition sequences used in RNAi constructs (black bars). 5’ and 3’ untranslated regions (UTRs) are marked by grey boxes, coding sequence (CDS) by black boxes, with exons as indicated. Positions are given relative to the translational start codon of each transcript. Four RNAi constructs were generated Two of these (ZmUbi and ZmUbi) targeted both CrLFY1 and CrLFY2 using conserved coding sequence amplified from CrLFY1 (ZmUbi) or CrLFY2 (ZmUbi). The two remaining constructs (ZmUbi and ZmUbi) incorporate target sequence amplified from the 3’UTR region of CrLFY1 and CrLFY2, respectively. The position of primers used in target sequence amplification are shown. Primer sequences are supplied in the Key Resources table.

Figure 6—figure supplement 5.

Genotyping PCR confirms the presence of CrLFY RNAi T-DNA in transgenic lines and the absence of the RNAi hairpin in no hairpin control lines.

PCR was performed on genomic DNA extracted from T1 sporophytes pre-selected for antibiotic resistance. The presence of the HPT cassette (A) was confirmed in all lines by PCR. The presence or absence of both arms of the RNAi hairpin (B, C) were confirmed in each line by PCR, and where sequenced the amplified products were as expected.

Table 1.

Summary of CrLFY RNAi transgenic lines and their phenotypic characterization.

Transgenic lines exhibited gametophytic developmental arrest and/or sporophyte shoot termination at varying stages of development. ‘+’ indicates that a particular line was phenotypically normal at the developmental stage indicated, ‘−’ indicates that development had arrested at or prior to this stage. In lines marked ‘+/-’ the stage at which developmental defects occurred varied between individuals within the line, and at least some arrested individuals were identified at the stage indicated. The five ZmUbi lines shown were generated from three rounds of transformation, the pairs of lines B16 and B19 and D2 and D4 potentially arising from the same transformation event. The no hairpin control lines NHC-2 (F3) and NHC-3 (F4) may similarly have arisen from a single transformation event. In all other cases, each transgenic line arose from a separate round of transformation and so must represent independent T-DNA insertions.

RNAi transgene	Line	Transfor-mation replicate	Gametophyte phase				Sporophyte phase
			Spore germin-ation	AC-based growth	Notch meristem-based growth	% arrested	Embryo	Shoot apex initiated	Simple frond	Complex frond	% arrested
ZmUBIpro::CrLFY1/2-i1	B16	1	+	-	-	99.86	+	+	-	-	<5%
ZmUBIpro::CrLFY1/2-i1	B19	1	+	-	-	50.00	+	+	+	-	<5%
ZmUBIpro::CrLFY1/2-i1	D13	2	+	-	-	99.80	+	+	-	-	<5%
ZmUBIpro::CrLFY1/2-i1	D2	3	+	+	+	0.00	+	+	-	-	<5%
ZmUBIpro::CrLFY1/2-i1	D4	3	+	+	+	0.00	+	+	+	+	<5%
ZmUBIpro::CrLFY1/2-i2	F9	4	+	-	-	0.00	+	+	-	-	<5%
ZmUBIpro::CrLFY1/2-i2	F14	5	-	-	-	100.00	-	-	-	-	0
ZmUBIpro::CrLFY1-i3	E8	6	+	+/-	+/-	100.00	-	-	-	-	0
ZmUBIpro::CrLFY1-i3	G13	7	+	+	+	0.00	+	+	-	-	<5%
ZmUBIpro::CrLFY2-i4	C3	8	+	+	+	0.00	+	+	-	-	<5%
NHC-1 (control)	D20	9	+	+	+	0.00	+	+	+	+	0
NHC-2 (control)	F3	10	+	+	+	0.00	+	+	+	+	0
NHC-3 (control)	F4	10	+	+	+	0.00	+	+	+	+	0

In the no hairpin control (NHC) plants, post-embryonic shoot development initiated with the production of simple, spade-like fronds from the shoot apex (Figure 6A) as in wild type. In eight transgenic lines, sub-populations of sporophytes developed in which this early stage of sporophyte development was perturbed, one line (E8) failing to initiate recognizable embryos (Figure 6B) and the remainder exhibiting premature shoot apex termination, typically after producing several distorted fronds (Figure 6C–H). Sub-populations of phenotypically normal transgenic sporophytes were also identified in some of these lines (Figure 6I–L). The two remaining lines exhibited less severe shoot phenotypes, one undergoing shoot termination after the production of simple (B19a) or lobed (B19b) fronds at the stage when control sporophytes produced complex dissected fronds (Figure 6M–O), and the other (D4b) completing sporophyte development but reduced in size to approximately 50% of controls (Figure 6P,Q). Despite the predicted sequence specificity of CrLFY1-i3 and CrLFY2-i4 (Supplementary file 5), qRT-PCR analysis found that all four RNAi constructs led to suppressed transcript levels of both CrLFY genes (Figure 6R,S). The severity of the shoot phenotype was correlated with the level of endogenous CrLFY transcripts detected across all lines (Figure 6R,S), with relative levels of both CrLFY1 and CrLFY2 significantly reduced compared to controls in all early-terminating sporophytes (E8, G13, C3, D2, D4a, D13, F9) (p<0.01 or less). In phenotypically normal transgenic siblings CrLFY2 transcript levels were not significantly lower than controls (indeed in line D2, levels were higher p<0.01) whereas CrLFY1 levels were significantly reduced (p<0.0001), as in arrested siblings. Together, these data indicate that CrLFY2 can compensate for some loss of CrLFY1, but at least 22% of CrLFY1 activity is required for normal development (line D4a pn, Figure 6J,S). It can thus be concluded that CrLFY1 and CrLFY2 act partially redundantly to maintain indeterminacy of the shoot apex in Ceratopteris, a role not found in the early divergent bryophyte P. patens, nor known to be retained in the majority of later diverging flowering plants.

Figure 6.

Suppression of CrLFY expression causes early termination of the Ceratopteris sporophyte shoot apex.

(A-L) Sporophyte phenotype 25 days after fertilization (DAF) in no hairpin control, NHC-1 (A) and transgenic lines carrying RNAi constructs against CrLFY1 (ZmUbi) (B, C), CrLFY2 (ZmUbi) (D) and both CrLFY1 and CrLFY2 (ZmUbi and ZmUbi) (E–L). In some lines, both aborted and phenotypically normal sporophytes were identified (compare E and I; F and J; G and K; H and L). The presence of the RNAi transgene in phenotypically normal sporophytes was validated by genotyping (Figure 6—figure supplement 5). Scale bars = 1 mm (A–H), 5 mm (I–L). (M–Q) Sporophyte phenotype of two no hairpin control (NHC-3 and NHC-1) (M, P) and two ZmUbi (N, O) lines at 63 (M–O) and 76 (P,Q) DAF. (R, S) qRT-PCR analysis of CrLFY1 and CrLFY2 transcript levels (normalized against the averaged expression of reference genes CrACTIN1 and CrTBP) in the sporophytes of the RNAi lines shown in (A–L). Transcript levels are depicted relative to no hairpin controls (NHC-1or −3), n = 3, error bars = standard error of the mean (SEM). CrLFY1 and CrLFY2 expression levels were significantly reduced compared to controls (p<0.01 or less) in all transgenic lines where sporophyte shoots undergo early termination (A–H), but in phenotypically normal (pn) sporophytes segregating in the same lines (I–L), only CrLFY1 transcript levels were reduced (p<0.0001). CrLFY2 transcript levels in pn sporophytes were not significantly lower than in controls. Asterisks denote level of significant difference from controls (**p<0.01, ***p<0.001; ****p<0.0001).

Schematic of CrLFY1 and CrLFY2 transcripts, showing recognition sequences used in RNAi constructs (black bars). 5’ and 3’ untranslated regions (UTRs) are marked by grey boxes, coding sequence (CDS) by black boxes, with exons as indicated. Positions are given relative to the translational start codon of each transcript. Four RNAi constructs were generated Two of these (ZmUbi and ZmUbi) targeted both CrLFY1 and CrLFY2 using conserved coding sequence amplified from CrLFY1 (ZmUbi) or CrLFY2 (ZmUbi). The two remaining constructs (ZmUbi and ZmUbi) incorporate target sequence amplified from the 3’UTR region of CrLFY1 and CrLFY2, respectively. The position of primers used in target sequence amplification are shown. Primer sequences are supplied in the Key Resources table.

Each RNAi construct carries inverted repeats of CrLFY-derived sequence (see Figure 6—figure supplement 1 and Supplementary file 5) to generate a hairpin bridged by a linker sequence derived from the GUS CDS (Miki and Shimamoto, 2004). The positions given are relative to the start of the maize ubiquitin promoter (ZmUbi) that is driving RNAi expression, and are shown for ZmUbi, with the length of the RNAi target sequence varying between the four different constructs (see Figure 6—figure supplement 1). The sites of hybridization against probes for the GUS linker and hygromycin resistance marker (HygR) are shown. The position of a SacI restriction site (used in gel blot analysis, see Figure 6—figure supplement 3) is indicated. No SacI sites are present in any CrLFY target sequence used. The primers used in probe amplification are given in the Key Resources Table.

ZmUbi lines E8 and G13 were regenerated from two separate bombardments and so are necessarily independent of one another. Genomic DNA was extracted from four T1 sporophytes (arising from the free fertilization of T1 gametophytes) within each line, digested with SpeI and separated on an electrophoresis gel. Gel blot analysis of both constructs was performed with probes hybridizing either to the GUS linker of the RNAi hairpin or to the hygromycin resistance (HygR) CDS (Figure 6—figure supplement 2). For each full-length ZmUbi T-DNA insertion present in the genome a single insert is predicted to result in unlinked hybridization fragments with minimum sizes of ~1 kb (HygR) or ~3.6 kb (GUS linker). Based on the hybridization fragments obtained, line E8 carries two full-length insertions of the CrLFY1 RNAi cassette and line G13 carries two full-length insertions plus two additional partial insertions.

(A) Generalized schematic of CrLFY RNAi T-DNA with the relative position of primers used in genotyping PCR (see Figure 6—figure supplement 5, Figure 7—figure supplement 1) marked. The primer binding sites are common to all four RNAi constructs, with the size of the hairpin-containing PCR products varying due to the insertion of different CrLFY target sequences. (B) Primer combinations for each genotyping reaction and expected PCR product sizes for each CrLFY RNAi construct. Primer sequences are listed in the Key Resources Table.

PCR was performed on genomic DNA extracted from T1 sporophytes pre-selected for antibiotic resistance. The presence of the HPT cassette (A) was confirmed in all lines by PCR. The presence or absence of both arms of the RNAi hairpin (B, C) were confirmed in each line by PCR, and where sequenced the amplified products were as expected.

Figure 6—figure supplement 3.

Gel blot analysis of ZmUbi T1 transgenic lines.

ZmUbi lines E8 and G13 were regenerated from two separate bombardments and so are necessarily independent of one another. Genomic DNA was extracted from four T1 sporophytes (arising from the free fertilization of T1 gametophytes) within each line, digested with SpeI and separated on an electrophoresis gel. Gel blot analysis of both constructs was performed with probes hybridizing either to the GUS linker of the RNAi hairpin or to the hygromycin resistance (HygR) CDS (Figure 6—figure supplement 2). For each full-length ZmUbi T-DNA insertion present in the genome a single insert is predicted to result in unlinked hybridization fragments with minimum sizes of ~1 kb (HygR) or ~3.6 kb (GUS linker). Based on the hybridization fragments obtained, line E8 carries two full-length insertions of the CrLFY1 RNAi cassette and line G13 carries two full-length insertions plus two additional partial insertions.

Figure 6—figure supplement 2.

Generalized schematic of CrLFY RNAi constructs.

Each RNAi construct carries inverted repeats of CrLFY-derived sequence (see Figure 6—figure supplement 1 and Supplementary file 5) to generate a hairpin bridged by a linker sequence derived from the GUS CDS (Miki and Shimamoto, 2004). The positions given are relative to the start of the maize ubiquitin promoter (ZmUbi) that is driving RNAi expression, and are shown for ZmUbi, with the length of the RNAi target sequence varying between the four different constructs (see Figure 6—figure supplement 1). The sites of hybridization against probes for the GUS linker and hygromycin resistance marker (HygR) are shown. The position of a SacI restriction site (used in gel blot analysis, see Figure 6—figure supplement 3) is indicated. No SacI sites are present in any CrLFY target sequence used. The primers used in probe amplification are given in the Key Resources Table.

Figure 7—figure supplement 1.

Gametophytes exhibiting developmental arrest were transgenic.

A. Generalized schematic of CrLFY RNAi T-DNA with the relative position of primers used in genotyping PCR marked. Primer sequences and expected PCR product sizes for each CrLFY RNAi construct are given in Figure 6—figure supplement 4. Genotyping PCR was conducted on DNA extracted from single gametophytes exhibiting developmental arrest at 10 DPS in three ZmUbi lines. DNA from all arrested individuals amplified positive bands for the two hairpin arms (pVec8F-PW64R and PW64F-pVec8R) and for the hygromycin resistance marker (HPTF-HPTR).

CrLFY promotes apical cell divisions in the gametophyte

In six of the RNAi lines that exhibited sporophyte developmental defects, it was notable that 50–99% of gametophytes arrested development prior to the sporophyte phase of the lifecycle (Table 1). This observation suggested that LFY plays a role in Ceratopteris gametophyte development, a function not previously demonstrated in either bryophytes or angiosperms. During wild-type development, the Ceratopteris gametophyte germinates from a single-celled haploid spore, establishing a single apical cell (AC) within the first few cell divisions (Figure 7A). Divisions of the AC go on to form a two-dimensional photosynthetic thallus in both the hermaphrodite, where a notch meristem takes on growth (Figure 7B), and male sexes (Figure 7C) (Banks, 1999). In contrast, the gametophytes from six RNAi lines (carrying either ZmUbi, ZmUbi or ZmUbi) exhibited developmental arrest (Figure 7D–J), which in five lines clearly related to a failure of AC activity. The point at which AC arrest occurred varied, in the most severe line occurring prior to or during AC specification (Figure 7D) and in others during AC-driven thallus proliferation (Figure 7E–I). Failure of AC activity was observed in both hermaphrodites (Figure 7E) and males (Figure 7H,I). The phenotypically least-severe line exhibited hermaphrodite developmental arrest only after AC activity had been replaced by the notch meristem (Figure 7J). A role for CrLFY in maintenance of gametophyte AC activity was supported by the detection of CrLFY transcripts in the AC and immediate daughter cells of wild-type gametophytes by in situ hybridization (Figure 7K–N). By contrast CrLFY transcripts were not detected in arrested ZmUbi lines (Figure 7O–R) in which the presence of the transgene was confirmed by genotyping of individual arrested gametophytes (Figure 7—figure supplement 1). CrLFY1 and CrLFY2 transcripts could not be clearly distinguished in situ due to sequence similarity (see Supplementary file 6), and hence the observed phenotypes could not be ascribed to a specific gene copy. However, these data support a role for at least one CrLFY homolog in AC maintenance during gametophyte development, and thus invoke a role for LFY in the regulation of apical activity in both the sporophyte and gametophyte phases of vascular plant development.

Figure 7.

Suppression of CrLFY expression causes early termination of the Ceratopteris gametophyte apical cell.

(A–C) In no hairpin control lines, the gametophyte established a triangular apical cell (ac) shortly after spore (sp) germination (A). Divisions of the apical cell established a photosynthetic thallus in both hermaphrodite and male gametophytes. At 10 days post spore sowing (DPS) both gametophyte sexes were approaching maturity, with the hermaphrodite (B) having formed a chordate shape from divisions at a lateral notch meristem (n) and having produced egg-containing archegonia (ar), sperm-containing antheridia (an), and rhizoids (rh). The male (C) had a more uniform shape with antheridia across the surface. These phenotypes were identical to wild-type. (D–J) When screened at 10–17 DPS, gametophytes from multiple RNAi lines (as indicated) exhibited developmental arrest, mostly associated with a failure of apical cell activity. Arrest occurred at various stages of development from failure to specify an apical cell, resulting in only a rhizoid being produced and no thallus (D) through subsequent thallus proliferation (E–I). Gametophyte development in one line progressed to initiation of the notch meristem but overall thallus size was severely reduced compared to wild-type (J). (K–R) In situ hybridization with antisense probes detected CrLFY transcripts in the apical cell and immediate daughter cells of wild-type gametophytes at 4 DPS (K, M). No corresponding signal was detected in controls hybridized with sense probes (L, N). In the arrested gametophytes of two ZmUbi lines CrLFY transcripts could not be detected (O–R), and transgene presence was confirmed (Figure 7—figure supplement 1). Scale bars = 100 μm.

A. Generalized schematic of CrLFY RNAi T-DNA with the relative position of primers used in genotyping PCR marked. Primer sequences and expected PCR product sizes for each CrLFY RNAi construct are given in Figure 6—figure supplement 4. Genotyping PCR was conducted on DNA extracted from single gametophytes exhibiting developmental arrest at 10 DPS in three ZmUbi lines. DNA from all arrested individuals amplified positive bands for the two hairpin arms (pVec8F-PW64R and PW64F-pVec8R) and for the hygromycin resistance marker (HPTF-HPTR).

Figure 6—figure supplement 4.

Binding site of CrLFY RNAi genotyping PCR primers.

(A) Generalized schematic of CrLFY RNAi T-DNA with the relative position of primers used in genotyping PCR (see Figure 6—figure supplement 5, Figure 7—figure supplement 1) marked. The primer binding sites are common to all four RNAi constructs, with the size of the hairpin-containing PCR products varying due to the insertion of different CrLFY target sequences. (B) Primer combinations for each genotyping reaction and expected PCR product sizes for each CrLFY RNAi construct. Primer sequences are listed in the Key Resources Table.

Discussion

The results reported here reveal a role for LFY in the maintenance of apical cell activity throughout gametophyte and sporophyte shoot development in Ceratopteris. During sporophyte development, qRT-PCR and transgenic reporter lines demonstrated that CrLFY1 is preferentially expressed in the shoot apex (whether formed during embryogenesis or de novo on fronds, and both before and after the reproductive transition); in emerging lateral organ (frond) primordia; and in pinnae and pinnules as they form on dissected fronds (Figures 3–5). Notably, active cell division is the main feature in all of these contexts. CrLFY2 transcript levels were more uniform throughout sporophyte shoot development, in both dividing tissues and expanded fronds (Figure 3), and expression has previously been reported in roots (Himi et al., 2001). Simultaneous suppression of CrLFY1 and CrLFY2 activity by RNAi resulted in developmental arrest of both gametophyte and sporophyte shoot apices, with any fronds produced before termination of the sporophyte apex exhibiting abnormal morphologies (Figures 6 and 7). The severity of phenotypic perturbations in sporophytes of transgenic lines correlated with combined CrLFY1 and CrLFY2 transcript levels, with wild-type levels of CrLFY2 able to fully compensate for up to a 70% reduction in CrLFY1 levels (Figure 6). The duplicate CrLFY genes therefore act at least partially redundantly during shoot development in Ceratopteris.

A function for LFY in gametophyte development has not previously been reported in any land plant species. In the moss P. patens, PpLFY1 and PpLFY2 are expressed in both the main and lateral apices of gametophytic leafy shoots but double loss-offunction mutants develop normally, indicating that LFY is not necessary for maintenance of apical cell activity in the gametophyte (Tanahashi et al., 2005). By contrast, loss of CrLFY expression from the gametophyte shoot apex results in loss of apical cell activity during thallus formation in Ceratopteris (Figure 7). The different DNA binding site preferences (and hence downstream target sequences) of PpLFY and CrLFY (Sayou et al., 2014) may be sufficient to explain the functional distinction in moss and fern gametophytes, but the conserved expression pattern is intriguing given that there should be no pressure to retain that pattern in P. patens in the absence of functional necessity. The thalloid gametophytes of the two other extant bryophyte lineages (liverworts and hornworts) resemble the fern gametophyte more closely than mosses (Ligrone et al., 2012), but LFY function in these contexts is not yet known. Overall the data are consistent with the hypothesis that in the last common ancestor of ferns and angiosperms, LFY functioned to promote cell proliferation in the thalloid gametophyte, a role that has been lost in angiosperms where gametophytes have no apical cell and are instead just few-celled determinate structures.

The range of reported roles for LFY in sporophyte development can be rationalized by hypothesizing three sequential changes in gene function during land plant evolution (Figure 8). First, the ancestral LFY function to promote early cell divisions in the embryo was retained in vascular plants after they diverged from the bryophytes, leading to conserved roles in P. patens (Tanahashi et al., 2005) and Ceratopteris (Figure 4). Second, within the vascular plants (preceding divergence of the ferns) this proliferative role expanded to maintain post-embryonic apical cell activity, and hence to enable indeterminate shoot growth. This is evidenced by CrLFY activity at the tips of shoots, fronds and pinnae (Figures 4–6), all of which develop from one or more apical cells (Hill, 2001; Hou and Hill, 2004). Whether fern fronds are homologous to shoots or to leaves in angiosperms is an area of debate (Tomescu, 2009; Vasco et al., 2013; Harrison and Morris, 2018), but there are angiosperm examples of LFY function in the vegetative SAM (Ahearn et al., 2001; Zhao et al., 2018), axillary meristems (Kanrar et al., 2008; Rao et al., 2008; Chahtane et al., 2013) and in actively dividing regions of compound leaves (Hofer et al., 1997; Molinero-Rosales et al., 1999; Champagne et al., 2007; Wang et al., 2008; Monniaux et al., 2017) indicating that a proliferative role in vegetative tissues has been retained in at least some angiosperm species. Consistent with the suggestion that the angiosperm floral meristem represents a modified vegetative meristem (Theiben et al., 2016), the third stage of LFY evolution could have been co-option and adaptation of this proliferation-promoting network into floral meristems, with subsequent restriction to just the flowering role in many species. This is consistent with multiple observations of LFY expression in both vegetative and reproductive shoots (developing cones) in gymnosperms (Mellerowicz et al., 1998; Mouradov et al., 1998; Shindo et al., 2001; Carlsbecker et al., 2004; Vázquez-Lobo et al., 2007; Carlsbecker et al., 2013; Moyroud et al., 2017) and suggests that pre-existing LFY-dependent vegetative gene networks might have been co-opted during the origin of specialized sporophyte reproductive axes in ancestral seed plants, prior to the divergence of angiosperms.

Figure 8.

Evolutionary trajectory of LFY function.

The phylogeny was reconstructed from selected LFY protein sequences representing all extant embryophyte lineages (as highlighted) and the algal sister-group. Coloured bars at the terminal branches represent different developmental functions of LFY determined from functional analysis in those species (see Supplementary file 8 for references). Coloured numbers indicate the putative points of origin of different functions inferred from available data points across the tree. 1, cell division within the sporophyte zygote; 2, maintenance of indeterminate cell fate in vegetative shoots through proliferation of one or more apical cells (AC); 2a, maintenance of indeterminate cell fate in vegetative lateral/axillary apices; 2b, maintenance of indeterminate cell fate in the margins of developing lateral organs (compound leaves); 3, specification of floral meristem identity (determinate shoot development producing modified lateral organs) and shoot transition to the reproductive phase; 3a, maintenance of indeterminate cell fate in inflorescence lateral/branch meristems (in place of floral meristem fate).

The proposed evolutionary trajectory for LFY function bears some resemblance to that seen for KNOX protein function. Class I KNOX genes are key regulators of indeterminacy in the vegetative shoot apical meristem of angiosperms (Gaillochet et al., 2015), and are required for compound leaf formation in both tomato and Cardamine hirsuta (Bar and Ori, 2015). In ferns, KNOX gene expression is observed both in the shoot apex and developing fronds (Sano et al., 2005; Ambrose and Vasco, 2016), and in P. patens the genes regulate cell division patterns in the determinate sporophyte (Sakakibara et al., 2008). It can thus be speculated that LFY and KNOX had overlapping functions in the sporophyte of the last common ancestor of land plants, but by the divergence of ancestral angiosperms from gymnosperms, KNOX genes had come to dominate in vegetative meristems whereas LFY became increasingly specialized for floral meristem function. Unlike LFY, however, there is not yet any evidence for KNOX function in the gametophyte of any land plant lineage, and thus if a pathway for regulating stem cell activity was co-opted from the gametophyte into the sporophyte, the LFY pathway is the more likely one.

Materials and methods

Plant materials and growth conditions

All experimental work was conducted using Ceratopteris richardii strain Hn-n (Warne and Hickok, 1987). Plant growth conditions for Ceratopteris transformation and DNA gel blot analysis of transgenic lines were as previously described (Plackett et al., 2015).

Phylogenetic analysis

A dataset of 99 aligned LFY protein sequences from a broad range of streptophytes was first retrieved from Sayou et al. (2014). The dataset was pruned and then supplemented with further sequences (Supplementary file 1) to enable trees to be inferred that would (i) provide a more balanced distribution across the major plant groups and (ii) infer fern relationships. Only a subset of available angiosperm sequences was retained (keeping both monocot and dicot representatives) but protein sequences from other angiosperm species where function has been defined through loss-of-function analyses were added from NCBI – Antirrhinum majus FLO AAA62574.1 (Coen et al., 1990), Pisum sativum UNI AAC49782.1 (Hofer et al., 1997), Cucumis sativus CsLFY XP_004138016.1 (Zhao et al., 2018), Medicago truncatula SGL1 AY928184 (Wang et al., 2008), Petunia hybrida ALF AAC49912.1 (Souer et al., 1998), Nicotiana tabacum NFL1 AAC48985.1 and NFL2 AAC48986.1 (Kelly, 1995), Eschscholzia californica EcFLO AAO49794.1 (Busch and Gleissberg, 2003), Gerbera hybrida cv. ‘Terraregina’ GhLFY ANS10152.1 (Zhao et al., 2016), Lotus japonicus LjLFY AAX13294.1 (Dong et al., 2005) and Populus trichocarpa PTLF AAB51533.1 (Rottmann et al., 2000). To provide better resolution within and between angiosperm clades, sequences from Spirodela polyrhiza (32G0007500), Zostera marina (27g00160.1), Aquilegia coerulea (5G327800.1) and Solanum tuberosum (PGSC0003DMT400036749) were added from Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html). Genome sequence from the early-diverging Eudicot Thalictrum thalictroides was searched by TBLASTX (Altschul et al., 1990) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastx&PAGE_TYPE=BlastSearch&BLAST_SPEC=&LINK_LOC=blasttab) with nucleotide sequence from the Arabidopsis LFY gene. A gene model was derived from sequence in two contigs (108877 and 116935) using Genewise (Birney et al., 2004) (https://www.ebi.ac.uk/Tools/psa/genewise/). Gymnosperm sequences were retained from Ginkgo biloba and from a subset of conifers included in Sayou et al. (2014), whilst sequences from conifers where in situ hybridization patterns have been reported were added from NCBI – Pinus radiata PRFLL AAB51587.1 and NLY AAB68601.1 (Mellerowicz et al., 1998; Mouradov et al., 1998) and Picea abies PaLFY AAV49504.1 and PaNLY AAV49503.1 (Carlsbecker et al., 2004). Fern sequences were retained except Angiopteris spp sequences which consistently disrupted the topology of the tree by grouping with gymnosperms. To better resolve relationships within the ferns, additional sequences were identified in both NCBI and 1KP (Matasci et al., 2014) databases. The protein sequence from Matteuccia struthiopteris AAF77608.1 MatstFLO (Himi et al., 2001) was retrieved from NCBI. Further sequences from horsetails (2), plus eusporangiate (1) and leptosporangiate (53) ferns were retrieved from the 1KP database (https://db.cngb.org/blast/) using BLASTP and the MatstFLO sequence as a query. Lycophyte and bryophyte sequences were all retained, but the liverwort Marchantia polymorpha predicted ORF sequence was updated from Phytozome v12.1 (Mpo0113s0034.1.p), the hornwort Nothoceros genome scaffold was replaced with a translated full length cDNA sequence (AHJ90704.1) from NCBI and two additional lycophyte sequences were added from the 1KP dataset (Isoetes tegetiformans scaffold 2013584 and Selaginella kraussiana scaffold 2008343). All of the charophyte scaffold sequences were substituted with Coleochaete scutata (AHJ90705.1) and Klebsormidium subtile (AHJ90707.1) translated full-length cDNAs from NCBI.

The new/replacement sequences were trimmed and amino acids aligned to the existing alignment from Sayou et al. (2014) using CLUSTALW (Li et al., 2015) (Supplementary file 2 and 3). The best-fitting model parameters (JTT + I + G4) were estimated and consensus phylogenetic trees were run using Maximum Likelihood from 1000 bootstrap replicates, using IQTREE (Nguyen et al., 2015). Two trees were inferred. The first contained only a subset of fern and allied sequences to achieve a more balanced distribution across the major plant groups (81 sequences in total) (Figure 8), whereas the second used the entire dataset (120 sequences ~ 50% of which are fern and allied sequences – Figure 1—figure supplement 1). The data were imported into ITOL (Letunic and Bork, 2016) to generate the pictorial representations. All branches with less than 50% bootstrap support were collapsed. Relationships within the ferns (Figure 1) were represented by pruning the lycophyte and fern sequences (68 in total) from the tree containing all available fern sequences (

CrLFY locus characterization and DNA gel blot analysis

Because no reference genome has yet been established for Ceratopteris (or any fern), CrLFY copy number was quantified by DNA gel blot analysis. Ceratopteris genomic DNA was hybridized using both the highly conserved LFY DNA-binding domain diagnostic of the LFY gene family (Maizel et al., 2005) and also gene copy-specific sequences (Figure 1—figure supplement 2). CrLFY1 and CrLFY2 share 85% amino acid similarity, compared to 65% and 44% similarity of each to AtLFY. DNA gel blotting and hybridization was performed as described previously (Plackett et al., 2014). The results supported the presence of only two copies of LFY within the Ceratopteris genome. All primers used in probe preparation are supplied in the Key Resources Table.

Genomic sequences for CrLFY1 and CrLFY2 open reading frames (ORFs) were amplified by PCR from wild-type genomic DNA using primers designed against published transcript sequences (Himi et al., 2001). ORFs of 1551 bp and 2108 bp were obtained, respectively (Figure 1—figure supplement 2). Exon structure was determined by comparison between genomic and transcript sequences. The native promoter region of CrLFY1 was amplified from genomic template by sequential rounds of inverse PCR with initial primer pairs designed against published CrLFY1 5’UTR sequence and additional primers subsequently designed against additional contiguous sequence that was retrieved. A 3.9 kb contiguous promoter fragment was isolated for CrLFY1 containing the entire published 5’UTR and 1.9 kb of additional upstream sequence (Figure 1—figure supplement 2). Repeated attempts were made to obtain a CrLFY2 promoter fragment but this proved impossible in the absence of a reference genome. Some sequence contiguous with the CrLFY2 ORF was obtained by inverse PCR using primers designed against the previously published 5’UTR sequence of the CrLFY2 transcript (Himi et al., 2001). This sequence was extended to 1016 bp in length using additional primers against the isolated genomic sequence but this fragment did not contain the entire published 5’UTR. Numerous rounds of inverse PCR generated a second 3619 bp genomic fragment containing sequence identical to the remaining 5’UTR (see Figure 1—figure supplement 2, Supplementary file 7) but the presumed connecting sequence between these two fragments could not be amplified despite many attempts. It was eventually concluded that either the intervening promoter fragment was too long to amplify or that it was too GC rich for amplification. All primers used in ORF amplification and inverse PCR are supplied in the Key Resources table. The contiguous sequences obtained for the CrLFY1 and CrLFY2 genomic loci have been submitted to Genbank (accessions MH841970 and MH841971, respectively).

qRT-PCR analysis of gene expression

RNA was extracted from Ceratopteris tissues using the Spectrum Total Plant RNA kit (Sigma-Aldrich, St. Louis, MO) and 480 ng were used as template in iScript cDNA synthesis (Bio-Rad). CrLFY1 and CrLFY2 locus-specific qRT-PCR primers were designed spanning intron 1. Amplification specificity of primers was validated via PCR followed by sequencing. qRT-PCR of three biological replicates and three technical replicates each was performed in a Bio-Rad CFX Connect with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). Primer amplification efficiency was checked with a cDNA serial dilution. Efficiency was determined using the slope of the linear regression line as calculated by Bio-Rad CFX Connect software. Primer specificity was tested via melting curve analysis, resulting in a single peak per primer set. CrLFY expression was calculated using the 2- ΔΔCt method (Livak and Schmittgen, 2001) and normalized against the geometric mean of the expression of two endogenous reference genes (Hellemans et al., 2007), CrACTIN1 and CrTATA-BINDING PROTEIN (TBP) (Ganger et al., 2015). The standard deviation of the Ct values of each reference gene was calculated to ensure minimal variation (<3%) in gene expression. Error bars represent ± the standard error of the mean of the 2 ΔΔ Ct values. All primers used in qRT-PCR are supplied in the Key Resources table.

Relative expression values of CrLFY from qRT-PCR were compared by one or two-way analysis of variance (ANOVA) for developmental stages followed by Tukey’s or Sidak’s multiple comparisons, respectively. To test whether genes were downregulated in transgenic RNAi lines, two-way ANOVA was perfomed with gene (CrLFY1 or CrLFY2) and transgenic line as factors, with ‘gene’ as a repeated factor when all transgenic lines had the same number of replicates. Where appropriate, expression of each gene in each line was compared to the expression of the respective control by Dunnet comparisons. Control plants had been transformed and were hygromycin-resistant, but did not contain the RNAi hairpin that triggers gene silencing (non-hairpin controls, NHC). For all experiments, NHCs were grown alongside transgenic lines. qRT-PCR of transgenic lines was necessarily conducted across several plates, each including a representative NHC, and statistical comparisons were performed within each plate relative to its respective control. The significance threshold (p) was set at 0.05. All statistical analyses were performed in Prism v. 6.0 (GraphPad Software, Inc., La Jolla, CA).

Generation of GUS reporter constructs

The CrLFY1 reporter construct (Figure 4—figure supplement 1) was created by cloning the CrLFY1 promoter into pART7 as a NotI-XbaI restriction fragment, replacing the existing 35S promoter. A β-Glucuronidase (GUS) coding sequence (Ulmasov, 1997) was cloned downstream of pCrLFY1 as an XbaI-XbaI fragment. The same GUS XbaI-XbaI fragment was also cloned into pART7 to create a 35S positive control (Figure 4—figure supplement 4). The resulting CrLFY1 and 35S cassettes were each cloned as NotI-NotI fragments into the pART27-based binary transformation vector pBOMBER carrying a hygromycin resistance marker previously optimized for Ceratopteris transformation (Plackett et al., 2015). All primers used in GUS reporter component amplification are supplied in the Key Resources table.

Generation of RNAi constructs

RNAi constructs were designed and constructed using the pANDA RNAi expression system (Miki and Shimamoto, 2004). Four RNAi fragments were designed, two targeting a conserved region of the CrLFY1 and CrLFY2 coding sequence (77% nucleotide identity) using sequences from either CrLFY1 (CrLFY1/2-i1) or CrLFY2 (CrLFY1/2-i2), and two targeting gene-specific sequence within the 3’UTR of CrLFY1 (CrLFY1-i3) or CrLFY2 (CrLFY2-i4) (Figure 6—figure supplement 1). Target fragments were amplified from cDNA and cloned into Gateway-compatible entry vector pDONR207 (Invitrogen, Carlsbad, CA). Each sequence was then recombined into the pANDA expression vector via Gateway LR cloning (Invitrogen, Carlsbad, CA). All primers used in RNAi target fragment amplification are supplied in the Key Resources table.

Generation of transgenic lines

Transformation of all transgenes into wild-type Hn-n Ceratopteris callus was performed as previously described (Plackett et al., 2015). T0 sporophyte shoots were regenerated from transformed callus tissue, with each round of transformation using multiple separate pieces of callus as starting material. Transgenic T1 spores were harvested from these T0 shoots, germinated to form T1 gametophytes and then self-fertilized to produce T1 sporophytes. T1 sporophytes were assessed for T-DNA copy number by DNA gel blot analysis (Figure 4—figure supplement 2; Figure 6—figure supplement 3) and the presence of full-length T-DNA insertions was confirmed through genotyping PCR (Figure 4—figure supplements 3 and 4). All primers used in genotyping reactions are supplied in the Key Resources table. For characterization of RNAi lines, T2 spores were collected from individuals that either contained the full transgene construct or from segregants in which the RNAi hairpin was absent.

Figure 4—figure supplement 3.

PCR analysis of CrLFY1 T1 lines identified full-length or near full-length CrLFY1 promoter sequences in T-DNA insertions.

(A) Schematic of the CrLFY1 construct marking binding sites of PCR primers used in (B). All positions are given relative to the GUS ATG. Primer sequences are listed in the Key Resources Table. (B) PCR was performed on genomic DNA from the same four individual T1 sporophytes within each line investigated by gel blot analysis (see Figure 4—figure supplement 2). PCR reactions were performed to amplify the native 3.9 kb CrLFY1 promoter as a positive control (row 1) and to amplify T-DNA specific products (rows 2–4) containing the GUS CDS and different lengths of contiguous CrLFY1 promoter sequence (see A). Black arrowheads mark the expected size of the target PCR product in each reaction. PCR analysis identified at least one full-length GUS CDS with ~3.8 kb of CrLFY1 promoter in line AF3 (row 2). Faint PCR products indicate that line AG18 and AE2 probably contain a full-length GUS CDS plus ~3 kb of CrLFY1 promoter sequence (row 3). All lines carry a GUS CDS plus a minimum CrLFY1 promoter length of 875 bp (row 4).

GUS staining

GUS activity analysis in CrLFY1 transgenic lines was conducted in the T1 generation. GUS staining was conducted as described previously (Plackett et al., 2014). Optimum staining conditions (1 mg/ml X-GlcA, 5 μM potassium ferricyanide) were determined empirically. Tissue was cleared with sequential incubations in 70% ethanol until no further decolorization occurred. GUS-stained gametophytes were imaged with a Zeiss Axioplan microscope and GUS-stained sporophytes imaged with a dissecting microscope, both mounted with Q-imaging Micro-published 3.3 RTV cameras. Images were minimally processed for brightness and contrast in Photoshop (CS4).

Phenotypic characterization

Phenotypic characterization of RNAi transgenic lines was conducted in the T2 or T3 generation. Isogenic lines were obtained by isolating hermaphrodite gametophytes in individual wells at approximately 7 DPS (or when the notch became visible, whichever came first) and flooding them once they had developed mature gametangia (at approximately 9 DPS). All transgenic lines were grown alongside both wild-type and no hairpin controls, and phenotypes observed and recorded daily. Gametophytes exhibiting altered phenotypes were imaged at approximately 10 DPS with a Nikon Microphot-FX microscope. Sporophytes with abnormal phenotypes were imaged with a dissecting microscope.

In situ hybridization

Antisense and sense RNA probes for CrLFY1 and CrLFY2 were amplified and cloned into pCR 4-TOPO (Invitrogen) and DIG-labelled according to the manufacturer’s instructions (Roche, Indianapolis, IN). Probes were designed to include the 5’UTR and ORF (CrLFY1 521 bp 5’UTR and 1113 bp ORF; CrLFY2 301 bp 5’UTR and 1185 bp ORF) (Supplementary file 6). All primers used in in situ probe amplification are supplied in the Key Resources table. We were unable to identify fragments that distinguished the two genes in whole mount in situ hybridizations. Tissue was fixed in FAA (3.7% formaldehyde, 5% acetic acid; 50% ethanol) for 1–4 hr and then stored in 70% ethanol. Whole mount in situ hybridization was carried out based on Hejátko et al. (2006), with the following modifications: hybridization and wash steps were carried out in 24-well plates with custom-made transfer baskets (0.5 mL microcentrifuge tubes and 30 µm nylon mesh, Small Parts Inc., Logansport, IN). Permeabilization and post-fixation steps were omitted depending on tissue type to avoid damaging fragile gametophytes, Acetic Anhydride (Sigma-Aldrich) and 0.5% Blocking Reagent (Roche) washing steps were added to decrease background staining, and tissue was hybridized at 45°C. Photos were taken under bright-field with a Q-imaging Micro-publisher 3.3 RTV camera mounted on a Nikon Microphot-FX microscope. Images were minimally processed for brightness and contrast in Photoshop (CS4).

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered for publication in eLife in its present form.

The reviewers agreed that CrLFY2 promoter analysis and/or in situ hybridization, as well as controls for the RNAi analysis, are needed. eLife recommends a "reject" decision if it is likely that additional experiments will take more than 2 months. We therefore collectively agreed that rejection was the correct decision – however, the reviewers were all enthusiastic about the work and would strongly support re-submission to eLife if the requested experiments were included.

Reviewer #1:

This manuscript is the first to examine gene function using reverse genetics in a stably transformed fern, which is very exciting. The authors focus on LEAFY, which has been well characterized in moss and Arabidopsis. While it was previously known that the two Ceratopteris LEAFY genes are expressed in gametophytes and sporophytes, this study examines more detailed expression patterns and addresses their functions in both Ceratopteris generations by RNAi. The topic is very interesting and important in understanding fern development and plant evolution in general.

Comments/questions relating to the gene expression studies:

1) Please describe the LFY promoter more; e.g., does it include all sequences upstream of the a) transcript initiation site b) the translation start site c) second exon that would include the first intron? This may be in the details of the supplementary figures (I couldn't tell), but I think it's important to include such details in the text.

2) Any reason why LFY2 promoter-GUS expression wasn't included? The conclusion that two LFY homologs in Ceratopteris function together to control of apical cell identity would be better supported if the expression patterns of CrLFY2 were examined using the same strategy (GUS reporter).

3) I assume that a T1 sporophyte is the product a self of a gametophyte that came from the spores of a transformed sporophyte that was selected then regenerated from bombarded callus. Is this correct?

My comments/questions relating to the RNAi lines:

1) What does phenotypic screening in the line "phenotypic screening identified 10 lines with similar developmental defects that were associated with reduced CrLFY expression" mean? My concern here is that the phenotypes described may be caused by the callus derived shoot regeneration process, which typically happens during callus regeneration in many different plant species. The authors need to carefully characterize the phenotypes of multiple individual transgenic plants together with their segregated siblings that do not contain the RNAi construct. I would also suggest performing vigorous genetic analyses to clearly define and confirm the phenotypes from multiple independent transgenic lines (i.e., backcrossing to or by wild type and examining whether the phenotype is tightly correlated to the repression of LFY genes in the F2 population). Wouldn't selection for drug resistance be useful here?

2) If some of the lines never develop gametophytes with gametangia, how were T2 or T3 lines generated?

My comments regarding the Discussion:

Much of this study is based on a single fern (Ceratopteris) and on one gene (LFY). For this reason, I regard the discussion of the evolutionary trajectory of LFY to be very speculative. The last sentence "if a pathway for regulating stem cell activity was co-opted from the gametophyte into the sporophyte, it was the LFY pathway" should be changed to.. if a pathway for regulating stem cell activity was co-opted from the gametophyte into the sporophyte, the pathway involving LFY is more likely than the KNOX pathway." There could be other pathways as well.

Because the Discussion focuses on evolution, it is important that this study include at least some supporting data from at least one other fern.

Reviewer #2:

Comparative analyses between bryophytes and angiosperms have been provided the information how gene function evolved to facilitate developmental innovations during land plant evolution as the authors pointed out. Ferns are one of missing link though it takes very important position during land plant evolution. Ceratopteris has been recognized as a model fern because of the early life cycle. The establishment of transformation technique in Ceratopteris became a breakthrough in this field (Muthukumar et al., 2013, Plant Physiol.; Plackett et al., 2014; Bui et al., 2015, BMC Res. Notes). LFY is one of well-studied genes among land plant lineages (e.g. Maizel et al., 2005; Sayou et al., 2014). Functional analysis of fern Ceratopteris LFY genes should be interesting topic for many readers.

The authors carefully examined the expression level of two Ceratopteris LFY genes, CrLFY1 and 2, spatial expression analysis of CrLFY1 using promoter analysis, and suppression analysis of CrLFY1 and CrLFY2 activity by RNAi. This paper will be one of model case of EvoDevo study using the fern Ceratopteris. The quality of analyses and figures is excellent, however two questions remain.

1) Why the authors did not provide the expression data by CrLFY2 promoter analysis nor in situ hybridization data of CrLFY2.

The authors indicated that CrLFY1 and CrLFY2 were differentially expressed during the Ceratopteris life cycle (subsection “CrLFY1 and CrLFY2 transcripts accumulate differentially during the Ceratopteris lifecycle”) and CrLFY1 and CrLFY2 act partially redundantly to maintain indeterminacy of the shoot apex in Ceratopteris (subsection “CrLFY1 regulates activity of the sporophyte shoot apex”). Also they discussed that the duplicate CrLFY genes therefore act at least partially redundantly during shoot development in Ceratopteris (Discussion, first paragraph). It would be very helpful to understand the difference and redundancy in the function of CrLFY1 and 2, if they have the spatial expression data of CrLFY2.

The authors described that no GUS activity was detected in unfertilized archegonia of CrLFY1 gametophytes (subsection “Spatial expression patterns of CrLFY1 are consistent with a retained ancestral role to facilitate cell divisions during embryogenesis”). In the case, the expression of CrLFY2 should to be important to function in the gametophytes. The spatial expression patterns of CrLFY2 would provide important information for the hypothesis. Since the authors succeeded both transformation and in situ hybridization in Ceratopteris, it seems technically possible to provide these data.

2) The authors provided that RNAi suppression analysis of CrLFY1 and CrLFY2 by using 4 different constructs and used wild type strain as control. The strain introduced with the vector is better to use as control. Transformation trials from three constructs produced a few transformants and the phenotype from same construct is different. The authors explained the difference of phenotype was because of the expression level of CrLFY1 or CrLFY2, though also should consider the effect of transformation procedure or transformation with the vector per se.

Reviewer #3:

This manuscript analyses the role of the LEAFY transcription factor in the fern Ceratopteris. The major finding is that LEAFY is expressed and essential for apical cell division both in the gametophyte and the sporophyte. LEAFY is a transcription factor playing a major role during flower development in angiosperms. It is present in most charophytes algae and all land plants. Its role has only been analyzed genetically in the moss Physcomitrella patens where it controls cell division in the sporophyte. In angiosperms, there is a growing body of evidence that LEAFY plays a role in meristem development (not necessarily flower meristem) as illustrated in various species such as rice, cucumber or pea. The evolutionary scenario describing how LEAFY could have been co-opted from a role in sporophyte cell division to flower development has remained very uncertain because of the lack of genetics in plant groups such as ferns and gymnosperms. In species belonging to these groups, one had to rely on expression patterns and inference on the function but there was, until now, no genetic evidence.

The work presented here represents a great leap forward by partially filling the gap between mosses and angiosperms. It analyses the expression and function of the LEAFY genes in Ceratopteris and concludes that LEAFY plays a role in apical growth in the sporophyte and gametophyte. To my opinion, these conclusions are well supported. Generating and analysing fern transgenics is a challenging but extremely useful task to learn about plant evolution in the major plant families. Even if the work is based mostly on expression pattern and mutant description (and it might look a bit old-fashioned at first glance for the lack of attempt to identify regulated genes), I am convinced that the results presented here are of great significance not only for the LEAFY research field but as an example of how a transcription factor can be co-opted along plant evolution to acquire a key role in angiosperms. Such a trajectory could not have been guessed without this type of seminal work in more basal plants.

My only concern is that this work is not so easy to follow for those who are not familiar with fern development. I suggest adding a scheme describing Ceraptopteris life cycle (and naming relevant organs and tissues) that can referred to as a guide for each figure showing expression patterns (GUS or mRNA) or phenotypes.

[Editors’ note: the author responses to the first round of peer review follow.]

Many thanks for the well-considered handling of our manuscript. The reviews and decision are perfectly understandable based on the contents of the submitted manuscript and I am grateful to the reviewers for their enthusiasm and informed critique. I am especially grateful for the acceptance that any further experiments would be impossible in two months. However, I attach a document that provides some extra information relating to the two main issues – CrLFY2 promoter analysis and RNAi controls. I have highlighted the main points where we can add information to clarify/justify our approach. I do believe that the conclusions we have made are robust and justifiable but accept that we could be more circumspect in the discussion.

In brief:

1) The CrLFY2 analysis is impossible until a genome sequence is available.

2) We chose wild-type as the standard control for RNAi lines because null segregants are rarely that after bombardment in Ceratopteris – each is likely to contain a fragment of the construct even if the hairpin is absent.

Reviewer #1:

[…] Comments/questions relating to the gene expression studies:

1) Please describe the LFY promoter more; e.g., does it include all sequences upstream of the a) transcript initiation site b) the translation start site c) second exon that would include the first intron? This may be in the details of the supplementary figures (I couldn't tell), but I think it's important to include such details in the text.

A diagrammatic representation of the promoter and 5’UTR structure was included as part of Figure 1—figure supplement 2 and referred to in the Materials and methods. Details are now also in the

text of the Results.

2) Any reason why LFY2 promoter-GUS expression wasn't included? The conclusion that two LFY homologs in Ceratopteris function together to control of apical cell identity would be better supported if the expression patterns of CrLFY2 were examined using the same strategy (GUS reporter).

It would have been ideal to include expression data for both genes and numerous attempts were made to obtain a CrLFY2 promoter fragment for use in GUS expression studies alongside CrLFY1, but this proved impossible in the absence of a reference genome. Details of what we attempted to do are now in the Materials and methods, and reference to the fact that we were unable to amplify a CrLFY2 promoter for use in GUS assays is now in the results text. We also refer to the fact that expression could not be determined in situ as we were unable to identify a fragment that clearly distinguished CrLFY1 and CrLFY2 in hybridizations (in main text and Materials and methods).

3) I assume that a T1 sporophyte is the product a self of a gametophyte that came from the spores of a transformed sporophyte that was selected then regenerated from bombarded callus. Is this correct?

This is now clearly articulated in the Materials and methods.

My comments/questions relating to the RNAi lines:

1) What does phenotypic screening in the line "phenotypic screening identified 10 lines with similar developmental defects that were associated with reduced CrLFY expression" mean? My concern here is that the phenotypes described may be caused by the callus derived shoot regeneration process, which typically happens during callus regeneration in many different plant species. The authors need to carefully characterize the phenotypes of multiple individual transgenic plants together with their segregated siblings that do not contain the RNAi construct.

We compared the phenotype of transgenic lines containing the full transgene with that of wild-type and of transgenic controls that lacked the RNAi hairpin (no hairpin controls – NHC). Wild type and NHC lines were phenotypically indistinguishable and NHC lines did not show suppression of endogenous LFY expression (see new Figure 6, Supplementary file 6, Table 1). Because DNA fragmentation after bombardment is common, these lines still contain part of the transgene cassette. Importantly, the new controls do not change the interpretation of any of the RNAi data.

I would also suggest performing vigorous genetic analyses to clearly define and confirm the phenotypes from multiple independent transgenic lines (i.e., backcrossing to or by wild type and examining whether the phenotype is tightly correlated to the repression of LFY genes in the F2 population). Wouldn't selection for drug resistance be useful here?

We did not do this routinely but because the selection antibiotic prevented fertilization of gametophytes, we did in some cases grow T1 gametophyte populations on non-selective medium and allow unrestricted mating to occur within lines which were determined to be a mixed population of transgenic and non-transgenic individuals (based on relative survival on and off antibiotic selection). Transgenic individuals were then identified post-fertilization by screening directly for antibiotic resistance. In these cases, T1 sporophytes would represent either hemizygous or homozygous individuals, some of the hemizygous being effectively backcrossed to wild-type. Gel-blot analysis found that sibling sporophytes generated within a line in this manner had a similar pattern of T-DNA insertions, implying very little genetic variation within each T1 population. There was also very little phenotypic variation between individuals.

2) If some of the lines never develop gametophytes with gametangia, how were T2 or T3 lines generated?

There were only two lines in which 100% of the gametophytes arrested (F14 and E8). It was thus possible to look at sporophyte development in the remaining 8 (7 of which are shown in new Figure 6).

My comments regarding the Discussion:

Much of this study is based on a single fern (Ceratopteris) and on one gene (LFY). For this reason, I regard the discussion of the evolutionary trajectory of LFY to be very speculative. The last sentence "if a pathway for regulating stem cell activity was co-opted from the gametophyte into the sporophyte, it was the LFY pathway" should be changed to.. if a pathway for regulating stem cell activity was co-opted from the gametophyte into the sporophyte, the pathway involving LFY is more likely than the KNOX pathway." There could be other pathways as well.

This sentence has been changed.

Because the Discussion focuses on evolution, it is important that this study include at least some supporting data from at least one other fern.

This is not feasible at this stage because of the paucity of other model fern systems, neither has it been a requirement in other evo-devo studies e.g., with mosses (focused on Physcomitrella patens) or liverworts (Marchantia polymorpha). The point of the Discussion is to set up a hypothesis that can be further validated in future studies.

Reviewer #2:

[…] The authors carefully examined the expression level of two Ceratopteris LFY genes, CrLFY1 and 2, spatial expression analysis of CrLFY1 using promoter analysis, and suppression analysis of CrLFY1 and CrLFY2 activity by RNAi. This paper will be one of model case of EvoDevo study using the fern Ceratopteris. The quality of analyses and figures is excellent, however two questions remain.

1) Why the authors did not provide the expression data by CrLFY2 promoter analysis nor in situ hybridization data of CrLFY2.

The authors indicated that CrLFY1 and CrLFY2 were differentially expressed during the Ceratopteris life cycle (subsection “CrLFY1 and CrLFY2 transcripts accumulate differentially during the Ceratopteris lifecycle”) and CrLFY1 and CrLFY2 act partially redundantly to maintain indeterminacy of the shoot apex in Ceratopteris (subsection “CrLFY1 regulates activity of the sporophyte shoot apex”). Also they discussed that the duplicate CrLFY genes therefore act at least partially redundantly during shoot development in Ceratopteris (Discussion, first paragraph). It would be very helpful to understand the difference and redundancy in the function of CrLFY1 and 2, if they have the spatial expression data of CrLFY2.

The authors described that no GUS activity was detected in unfertilized archegonia of CrLFY1pro::GUS gametophytes (subsection “Spatial expression patterns of CrLFY1 are consistent with a retained ancestral role to facilitate cell divisions during embryogenesis”). In the case, the expression of CrLFY2 should to be important to function in the gametophytes. The spatial expression patterns of CrLFY2 would provide important information for the hypothesis. Since the authors succeeded both transformation and in situ hybridization in Ceratopteris, it seems technically possible to provide these data.

See response 2 to reviewer 1.

2) The authors provided that RNAi suppression analysis of CrLFY1 and CrLFY2 by using 4 different constructs and used wild type strain as control. The strain introduced with the vector is better to use as control. Transformation trials from three constructs produced a few transformants and the phenotype from same construct is different. The authors explained the difference of phenotype was because of the expression level of CrLFY1 or CrLFY2, though also should consider the effect of transformation procedure or transformation with the vector per se.

See response 4 to reviewer 1.

Reviewer #3:

[…] My only concern is that this work is not so easy to follow for those who are not familiar with fern development. I suggest adding a scheme describing Ceraptopteris life cycle (and naming relevant organs and tissues) that can referred to as a guide for each figure showing expression patterns (GUS or mRNA) or phenotypes.

We have inserted a schematic of the lifecycle showing morphology at different stages as new Figure 2. Citations of studies describing details of Ceratopteris development and morphology have now also been included in the text where appropriate.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Ceratopteris richardii)	CrLEAFY1 (CrLFY1)	Himi et al. (2001), PMID:11675598; This paper	NCBI:AB049974.2; NCBI:MH841970	cDNA only; ORF plus contiguous promoter
Gene (C. richardii)	CrLEAFY2 (CrLFY2)	Himi et al. (2001), PMID:11675598; This paper	NCBI:AB049975.2; NCBI:MH841971	cDNA only; ORF plus contiguous promoter
Strain, strain background (C. richardii)	Wild type (Hn-n)	Warne and Hickok, 1987, PMID:16665325
Genetic reagent (C. richardii)	CrLFY1/2-i1	This paper		C. richardii transgenic line; RNAi knockdown of CrLFY1 and CrLFY2 expression.
Genetic reagent (C. richardii)	CrLFY1/2-i2	This paper		C. richardii transgenic line; RNAi knockdown of CrLFY1 and CrLFY2 expression.
Genetic reagent (C. richardii)	CrLFY1-i3	This paper		C. richardii transgenic line; RNAi knockdown of CrLFY1 expression.
Genetic reagent (C. richardii)	CrLFY2-i4	This paper		C. richardii transgenic line; RNAi knockdown of CrLFY2 expression.
Genetic reagent (C. richardii)	CrLFY1pro::GUS	This paper		C. richardii transgenic line; CrLFY1pro::GUS reporter.
Genetic reagent (C. richardii)	35Spro::GUS	This paper		C. richardii transgenic line; 35Spro::GUS reporter.
Recombinant DNA reagent	CrLFY1pro	This paper	NCBI:MH841970	CrLFY1 5' genomic fragment; Figure 1—figure supplement 2
Recombinant DNA reagent	CrLFY2pro fragment 1	This paper	NCBI:MH841971	CrLFY2 5' genomic fragment; Figure 1—figure supplement 2
Recombinant DNA reagent	CrLFY2pro fragment 2	This paper		CrLFY2 5' genomic fragment; Figure 1—figure supplement 2; Supplementary file 7
Recombinant DNA reagent	GUS	Ulmasov, 1997, PMID:9401121		Β-Glucuronidase (GUS) coding sequence
Recombinant DNA reagent	pANDA (RNAi vector)	Miki and Shimamoto (2004), PMID:15111724
Recombinant DNA reagent	pCR4-TOPO (Cloning vector)	Invitrogen	Thermo Scientific: K457502
Recombinant DNA reagent	pDONR207 (Gateway vector)	Invitrogen
Recombinant DNA reagent	pBOMBER (Binary vector)	Plackett et al. (2015), PMID:26146510	NCBI:MH841969	Modified pART27 (PMID:1463857); Hygromycin resistance antibiotic selection marker
Recombinant DNA reagent	pART7 (Cloning vector)	Gleave 1992, PMID:1463857
Recombinant DNA reagent	ZmUbipro::CrLFY1/2-i1-pANDA	This paper		RNAi construct targeting CrLFY1 and CrLFY2; Figure 6—figure supplement 1; Figure 6—figure supplement 2
Recombinant DNA reagent	ZmUbipro::CrLFY1/2-i2-pANDA	This paper		RNAi construct targeting CrLFY1 and CrLFY2; Figure 6—figure supplement 1; Figure 6—figure supplement 2
Recombinant DNA reagent	ZmUbipro::CrLFY1-i3-pANDA	This paper		RNAi construct targeting CrLFY1; Figure 6—figure supplement 1; Figure 6—figure supplement 2
Recombinant DNA reagent	ZmUbipro::CrLFY2-i4-pANDA	This paper		RNAi construct targeting CrLFY2; Figure 6—figure supplement 1; Figure 6—figure supplement 2
Recombinant DNA reagent	CrLFY1pro::GUS-pBOMBER	This paper		GUS reporter construct, CrLFY1; Figure 4—figure supplement 1
Recombinant DNA reagent	35Spro::GUS-pBOMBER	This paper		GUS reporter construct, 35S control; Figure 4—figure supplement 1
Recombinant DNA reagent	CrLFY1 in situ probe (antisense)	This paper		In situ hybridisation probe; Supplementary file 6
Recombinant DNA reagent	CrLFY1 in situ probe (sense)	This paper		In situ hybridisation probe; Supplementary file 6
Recombinant DNA reagent	CrLFY2 in situ probe (antisense)	This paper		In situ hybridisation probe; Supplementary file 6
Recombinant DNA reagent	CrLFY2 in situ probe (sense)	This paper		In situ hybridisation probe; Supplementary file 6
Recombinant DNA reagent	32P-CrLFY1 probe 1	This paper		DNA gel blot probe for CrLFY1; Figure 1—figure supplement 2
Recombinant DNA reagent	32P-CrLFY1 probe 2	This paper		DNA gel blot probe for CrLFY1; Figure 1—figure supplement 2
Recombinant DNA reagent	32P-CrLFY2 probe 1	This paper		DNA gel blot probe for CrLFY2; Figure 1—figure supplement 2
Recombinant DNA reagent	32P-CrLFY2 probe 1	This paper		DNA gel blot probe for CrLFY2; Figure 1—figure supplement 2
Recombinant DNA reagent	32P-HygR probe	Plackett et al. (2014), PMID:24623851		DNA gel blot probe, T-DNA specific; Figure 4—figure supplement 1
Recombinant DNA reagent	32P-GUS probe	Plackett et al. (2014), PMID:24623851		DNA gel blot probe, T-DNA specific; Figure 4—figure supplement 1
Recombinant DNA reagent	32P-GUSlinker probe	This paper		DNA gel blot probe, T-DNA specific; Figure 6—figure supplement 2
Sequence-based reagent	CrLFY1ampF	This paper		ORF amplification, CrLFY1: 5'-ATGGATGTCTCT TTATTGCCAC-3'
Sequence-based reagent	CrLFY1ampR	This paper		ORF amplification, CrLFY1: 5'-TCAATCATAGATGC AGCTATCACTG-3'
Sequence-based reagent	CrLFY1ampF	This paper		ORF amplification, CrLFY2: 5'-ATGTTCCGATGG GAACAAAG-3'
Sequence-based reagent	CrLFY1ampR	This paper		ORF amplification, CrLFY2: 5'-TTATTCATAGCT GCAGCTGTC-3'
Sequence-based reagent	CrLFY1invF	This paper		Inverse PCR, CrLFY1: 5'-CTATGGAGTAC GAAGCACCAC-3'
Sequence-based reagent	CrLFY1invF2	This paper		Inverse PCR, CrLFY1: 5'-CGATCATTTCTT GTACTGCTCTC-3'
Sequence-based reagent	CrLFY1invF3	This paper		Inverse PCR, CrLFY1 : 5'-CAGTGCATGACCTTCGATATTG-3'
Sequence-based reagent	CrLFY1invR	This paper		Inverse PCR, CrLFY1: 5'-CAGTTGTTTCGGATCTGCAG-3'
Sequence-based reagent	CrLFY1invR2	This paper		Inverse PCR, CrLFY1: 5'-CTCCGCTTTTCATTTGAGAACG-3'
Sequence-based reagent	CrLFY1invR3	This paper		Inverse PCR, CrLFY1: 5'-CAAGAACCGCTGGAGTAAAC-3'
Sequence-based reagent	CrLFY2invF	This paper		Inverse PCR, CrLFY2: 5'-CTATGGTGTACGGAGCACTAC-3'
Sequence-based reagent	CrLFY2invF2	This paper		Inverse PCR, CrLFY2: 5'-CGTATCCAAAACAGC TTAAACTCC-3'
Sequence-based reagent	CrLFY2invF3	This paper		Inverse PCR, CrLFY2: 5'-CACTAAAGGTGCTGCTATCAAC-3'
Sequence-based reagent	CrLFY2invF4	This paper		Inverse PCR, CrLFY2: 5'-CATTGTGCTGACCTTGTGAAG-3'
Sequence-based reagent	CrLFY2invF5	This paper		Inverse PCR, CrLFY2: 5'-CGCAAAGGTTGGAA AAGAGAAC-3'
Sequence-based reagent	CrLFY2invF6	This paper		Inverse PCR, CrLFY2: 5'-CGACAACGGATCATAACCATC-3'
Sequence-based reagent	CrLFY2 invF7	This paper		Inverse PCR, CrLFY2: 5'-CAATAGTAGATT CTCCCTCCTTTAC-3'
Sequence-based reagent	CrLFY2invF8	This paper		Inverse PCR, CrLFY2: 5'-GCTCTTTAATTT GAATCACGTGTG-3'
Sequence-based reagent	CrLFY2invF9	This paper		Inverse PCR, CrLFY2: 5'-GAACAATGTGCA TGCGACTC-3'
Sequence-based reagent	CrLFY2invF10	This paper		Inverse PCR, CrLFY2: 5'-CATGTTCCGAT GGGAACAAAG-3'
Sequence-based reagent	CrLFY2invF11	This paper		Inverse PCR, CrLFY2: 5'-CATAGGGAACT CTGTAATGATGC-3'
Sequence-based reagent	CrLFY2invF12	This paper		Inverse PCR, CrLFY2: 5'-GTTTCCAG ATACTGCTGCTC-3'
Sequence-based reagent	CrLFY2invF13	This paper		Inverse PCR, CrLFY2: 5'-CATAGATGA TGCCAGTATACTCC-3'
Sequence-based reagent	CrLFY2invF14	This paper		Inverse PCR, CrLFY2: 5'-GCTCACTAT CCACAATTCATACAC-3'
Sequence-based reagent	CrLFY2invF15	This paper		Inverse PCR, CrLFY2: 5'-GTTCGTATCT GATACTTGTTTCGTG-3'
Sequence-based reagent	CrLFY2invF16	This paper		Inverse PCR, CrLFY2: 5'-CTTACTCCA CGAATGCATGC-3'
Sequence-based reagent	CrLFY2invR	This paper		Inverse PCR, CrLFY2: 5'-CAGTTGTCAC AGAGGTAGCAG-3'
Sequence-based reagent	CrLFY2invR2	This paper		Inverse PCR, CrLFY2: 5'-CCTTACGATG TATTACCCTTTGTTC-3'
Sequence-based reagent	CrLFY2invR3	This paper		Inverse PCR, CrLFY2: 5'-CAGTGACTA GGATGTCTGATACAG-3'
Sequence-based reagent	CrLFY2invR4	This paper		Inverse PCR, CrLFY2: 5'-GAAGGAGCT GAAAATGCAACTC-3'
Sequence-based reagent	CrLFY2invR5	This paper		Inverse PCR, CrLFY2: 5'-CCTGCCTCC TATGAAAACAC-3'
Sequence-based reagent	CrLFY2invR6	This paper		Inverse PCR, CrLFY2: 5'-CCTGTAAAGG AGGGAGAATCTAC-3'
Sequence-based reagent	CrLFY2invR7	This paper		Inverse PCR, CrLFY2: 5'-GCACTCCAAC GATGATGATAC-3'
Sequence-based reagent	CrLFY2invR8	This paper		Inverse PCR, CrLFY2: 5'-GCTGTACTA AGGCATCAATTCAG-3'
Sequence-based reagent	CrLFY2invR9	This paper		Inverse PCR, CrLFY2: 5'-CATCTATGATA GCACAACATCACTC-3'
Sequence-based reagent	CrLFY2invR10	This paper		Inverse PCR, CrLFY2: 5'-CACAACATC ACTCAGGACTC-3'
Sequence-based reagent	CrLFY2invR11	This paper		Inverse PCR, CrLFY2: 5'-CTGCCTCCTA TGAAAACACAAG-3'
Sequence-based reagent	CrLFY2invR12	This paper		Inverse PCR, CrLFY2: 5'-CTAGTCTTTG ATGAGGTTTCATGTC-3'
Sequence-based reagent	CrLFY2invR13	This paper		Inverse PCR, CrLFY2: 5'-CATGCAAGA AGCATGCAATTC-3'
Sequence-based reagent	CrLFY2invR14	This paper		Inverse PCR, CrLFY2: 5'-GTGTCTCCA GTAAGTATGAAACAAG-3'
Sequence-based reagent	CrLFY2invR15	This paper		Inverse PCR, CrLFY2: 5'-CATGAGGCC GTCAGACTTAC-3'
Sequence-based reagent	CrLFY2invR16	This paper		Inverse PCR, CrLFY2: 5'-CGTAACAGA CGAGCTCGATATAATAG-3'
Sequence-based reagent	CrLFY2invR17	This paper		Inverse PCR, CrLFY2: 5'-CTCTTTGCTCA TATAGCTTCAAGC-3'
Sequence-based reagent	CrLFY1 + 2 (1)-RNAi-F	This paper		T-DNA cloning, CrLFY1/2-i1: 5'-ATGGGT TTCACTGTGAATAC-3'
Sequence-based reagent	CrLFY1 + 2 (1)-RNAi-R	This paper		T-DNA cloning, CrLFY1/2-i1: 5'-TCTCCTC TTTGTTCCCTTGTG-3'
Sequence-based reagent	CrLFY1 + 2 (2)-RNAi-F	This paper		T-DNA cloning, CrLFY1/2-i2: 5'-ATGGG TTTCACTGTTAGTAC-3'
Sequence-based reagent	CrLFY1 + 2 (2)-RNAi-R	This paper		T-DNA cloning, CrLFY1/2-i2: 5'-TCTCCT CTTTGTTCCCTGGTG-3'
Sequence-based reagent	CrLFY1-RNAi-F	This paper		T-DNA cloning, CrLFY1-i3: 5'-CCTTTTCT TGCTAATGATGGC-3'
Sequence-based reagent	CrLFY1-RNAi-R	This paper		T-DNA cloning, CrLFY1-i3: 5'-CAAACAAA CTTGAAAATGATAC-3'
Sequence-based reagent	CrLFY2-RNAi-F	This paper		T-DNA cloning, CrLFY2-i4: 5'-GCCATTG CTAGCAAGGTTAT-3'
Sequence-based reagent	CrLFY2-RNAi-R	This paper		T-DNA cloning, CrLFY2-i4: 5'-CACTGCT TTGAAACTAAAAC-3'
Sequence-based reagent	pCrLFY1amp-NotF	This paper		T-DNA cloning, CrLFY1pro: 5'-CAGCGGCCGCTTAGATGG CTTGAGATGCTAC-3'
Sequence-based reagent	pCrLFY1amp-XbaR	This paper		T-DNA cloning, CrLFY1pro: 5'-CATCTAGAG GAGGCACTTCTTTACGTG-3'
Sequence-based reagent	GUSamp-XbaF	This paper		T-DNA cloning, GUS CDS: 5'-CATCTAGAC AATGGTAAGCTTAGCGGG-3'
Sequence-based reagent	GUSamp-XbaR	This paper		T-DNA cloning, GUS CDS: 5'-CCATCTAGA TTCATTGTTTGCCTCCCTG-3'
Sequence-based reagent	qCrLFY1_F2	This paper		qRT-PCR, CrLFY1: 5'-GTCCGCT ATTCGTGCAGAGA-3'
Sequence-based reagent	qCrLFY1_R2	This paper		qRT-PCR, CrLFY1 : 5'-AATTCAAGGGGG CATTGGGT-3'
Sequence-based reagent	qCrLFY2_F3	This paper		qRT-PCR, CrLFY2: 5'-GCAGTGACAATGAAGGACGC-3'
Sequence-based reagent	qCrLFY2_R3	This paper		qRT-PCR, CrLFY2: 5'-AGAATCGTGCACACTGCTCA-3'
Sequence-based reagent	qCrTBPb_F	Ganger et al. (2015), DOI:10.1139/cjb-2014–0202		qRT-PCR, CrTBP: 5'-ATGAGCCAGAGCTTTTCCCC-3'
Sequence-based reagent	qCrTBPb_R	Ganger et al. (2015), DOI:10.1139/cjb-2014–0202		qRT-PCR, CrTBP: 5'-TTCGTCTCTGACCTTTGCCC-3'
Sequence-based reagent	qCrACT1_F	Ganger et al. (2015), DOI:10.1139/cjb-2014–0202		qRT-PCR, CrActin1: 5'-GAGAGAGGCTA CTCTTTCACAACC-3'
Sequence-based reagent	qCrACT1_R	Ganger et al. (2015), DOI:10.1139/cjb-2014–0202		qRT-PCR, CrActin1: 5'-AGGAAGTTCGTA ACTCTTCTCCAA-3'
Sequence-based reagent	CrLFY1_ISH_F	This paper		In situ probes, CrLFY1: 5'-GAGGCATACA CACACGCAGT-3'
Sequence-based reagent	CrLFY1_ISH_R	This paper		In situ probes, CrLFY1: 5'-TCAATCATAGAT GCAGCTATCACTG-3
Sequence-based reagent	CrLFY2_ISH_F	This paper		In situ probes, CrLFY2: 5'-GGCTGGTTGTTA CGGATAGC-3'
Sequence-based reagent	CrLFY2_ISH_R	This paper		In situ probes, CrLFY2: 5'-TTATTCATAG CTGCAGCTGTCACTG-3'
Sequence-based reagent	CrLFY1_Probe1F	This paper		Copy number analysis, CrLFY1 probe 1: 5'-CAGG CACAAGGGAACAAAG-3'
Sequence-based reagent	CrLFY1_Probe1R	This paper		Copy number analysis, CrLFY1 probe 1: 5'-CA TAGATGCAGCTATCACTGTC-3'
Sequence-based reagent	CrLFY1_Probe2F	This paper		Copy number analysis, CrLFY1 probe 2: 5'-CACTTGAAGGTAAGCT TTATTGTAAGG-3'
Sequence-based reagent	CrLFY1_Probe2R	This paper		Copy number analysis, CrLFY1 probe 2: 5'-CAATA TTTCCGACTATACATTGAGGC-3'
Sequence-based reagent	CrLFY2_Probe1F	This paper		Copy number analysis, CrLFY2 probe 1: 5'-CAGGCA CCAGGGAACAAAG-3'
Sequence-based reagent	CrLFY2_Probe1R	This paper		Copy number analysis, CrLFY2 probe 1: 5'-CATAGC TGCAGCTGGTCACTGTC-3'
Sequence-based reagent	CrLFY2_Probe2F	This paper		Copy number analysis, CrLFY2 probe 2: 5'-CTGTAG AAGGTAAGATTCTGCTC-3'
Sequence-based reagent	CrLFY2_Probe2R	This paper		Copy number analysis, CrLFY2 probe 2: 5'-GCTT ATGGTACAGAATAAGTAGAGG-3'
Sequence-based reagent	HygF2	Plackett et al. (2014), PMID:24623851		T-DNA gel blot probe, HygR: 5'-CTTCTACA CAGCCATCGGTC-3'
Sequence-based reagent	HygR	Plackett et al. (2014), PMID:24623851		T-DNA gel blot probe, HygR: 5'-CCGATGGT TTCTACAAAGATCG-3'
Sequence-based reagent	GH3seqF3	Plackett et al. (2014), PMID:24623851		T-DNA gel blot probe, GUS: 5'-CTTCGCT GTACAGTTCTTTCG-3'
Sequence-based reagent	GH3seqR4	Plackett et al. (2014), PMID:24623851		T-DNA gel blot probe, GUS: 5'-CACTCATT ACGGCAAAGTGTG-3'
Sequence-based reagent	GUSlinkerseqF	This paper		T-DNA gel blot probe, RNAi: 5'-CTGATT AACCACAAACCGTTCTAC-3'
Sequence-based reagent	GUSlinkerseqR	This paper		T-DNA gel blot probe, RNAi: 5'-CTGATA CTCTTCACTCCACATG-3'
Sequence-based reagent	HPT-F	Miki and Shimamoto (2004), PMID:15111724		RNAi genotyping, HygR: 5'-GAGCCTGACCTA TTGCATCTCC-3'
Sequence-based reagent	HPT-R	Miki and Shimamoto (2004), PMID:15111724		RNAi genotyping, HygR: 5'-GGCCTCCAG AAGAAGATGTTGG-3'
Sequence-based reagent	pVec8F	Miki and Shimamoto (2004), PMID:15111724		RNAi genotyping, RNAi hairpin: 5'-TTTAGC CCTGCCTTCATACG-3'
Sequence-based reagent	pVec8R	Miki and Shimamoto (2004), PMID:15111724		RNAi genotyping, RNAi hairpin: 5'-ATTGC CAAATGTTTGAACGA-3'
Sequence-based reagent	PW64F	This paper		RNAi genotyping, RNAi hairpin: 5'-CATGAA GATGCGGACTTACG-3'
Sequence-based reagent	PW64R	This paper		RNAi genotyping, RNAi hairpin: 5'-ATCCAC GCCGTATTCGG-3'
Sequence-based reagent	pCrLFY1genoF1	This paper		CrLFY1pro::GUS genotyping: 5'-CTTAGA TGGCTTGAGATGCTAC-3'
Sequence-based reagent	pCrLFY1genoF2	This paper		CrLFY1pro::GUS genotyping: 5'-CTCTCT TCTTGCTTGTGTTGTG-3'
Sequence-based reagent	pCrLFY1genoF3	This paper		CrLFY1pro::GUS genotyping: 5'-CAACTGGCAACAGGTGATG-3'
Sequence-based reagent	pCrLFY1genoF4	This paper		CrLFY1pro::GUS genotyping: 5'-CAGTCTTAGTTCAACTGCATTCG-3'
Sequence-based reagent	pCrLFY1genoR	This paper		CrLFY1pro::GUS genotyping: 5'-AGGAGGCACTTCTTTACGTG-3'
Sequence-based reagent	GUSgenoR	This paper		CrLFY1pro::GUS + 35Spro::GUS genotyping: 5'-CATTGTTTG CCTCCCTGC-3'
Sequence-based reagent	35SgenoF	This paper		35Spro::GUS genotyping: 5'-CTGAGCTTAACAGCACAGTTG-3'
Sequence-based reagent	OCS3’genoR	This paper		35Spro::GUS genotyping: 5'-CATCACTAGTAAGCTAGCTTGC-3'
Commercial assay or kit	Phusion high-fidelity polymerase	Thermo Scientific	Thermo Scientific: F530S
Commercial assay or kit	Gateway LR clonase II enzyme mix	Invitrogen	Thermo Scientific: 11791100
Commercial assay or kit	QIAGEN Plasmid Maxi Kit	QIAGEN	QIAGEN:12163
Commercial assay or kit	Whatman Nytran nylon blotting membrane	GE Healthcare	GE Healthcare: 10416294
Commercial assay or kit	Random Primers DNA Labelling kit	Invitrogen	Thermo Scientific: 18187013
Commercial assay or kit	Carestream Kodak autoradiography GBX developer and fixer	Sigma-Aldrich	Sigma-Aldrich: Z354147
Commercial assay or kit	Carestream Kodak Biomax XAR film	Sigma-Aldrich	Sigma-Aldrich:F5763
Commercial assay or kit	iTaq universal SYBR Green mastermix	Bio-Rad	Bio-Rad:1725120
Commercial assay or kit	DIG-labelling mix	Roche Applied Sciences	Roche:11277073910
Commercial assay or kit	T3 RNA polymerase	Roche Applied Sciences	Roche:11031163001
Commercial assay or kit	T7 RNA polymerase	Roche Applied Sciences	Roche:10881767001
Commercial assay or kit	Anti-DIG antibody	Roche Applied Sciences	Roche:11093274910; RRID:AB_2313639
Commercial assay or kit	NBT/BCIP stock solution	Roche Applied Sciences	Roche:11681451001
Chemical compound, drug	Potassium ferricyanide (K₃Fe(CN)₆)	Sigma-Aldrich	Sigma:P8131
Chemical compound, drug	X-GlcA (CHA salt)	Melford Scientific	Melford:MB1021
Chemical compound, drug	CTP, [ɑ-32P]	Perkin Elmer	Perkin Elmer: BLU008H250UC
Software, algorithm	IQ-TREE	Nguyen et al. (2015), PMID:25371430		http://www.iqtree.org/
Software, algorithm	iTOL	Letunic and Bork (2016), PMID:27095192		https://itol.embl.de/
Software, algorithm	ClustalW	Li et al. (2015), PMID:25845596	RRID:SCR_002909	https://www.ebi.ac.uk/Tools/msa/clustalw2/
Software, algorithm	TBLASTX	Altschul et al. (1990), PMID:2231712	RRID:SCR_011823	https://blast.ncbi.nlm.nih.gov/Blast.cgi
Software, algorithm	GeneWise	Birney et al. (2004), PMID:15123596	RRID:SCR_015054	https://www.ebi.ac.uk/Tools/psa/genewise
Software, algorithm	GraphPad Prism	GraphPad Software Inc.	RRID:SCR_002798	https://www.graphpad.com/scientific-software/prism/
Software, algorithm	Adobe Photoshop CS4	Adobe	RRID:SCR_014199
Other	Biolistic PDS-1000/He Particle Delivery System	Bio-Rad	Bio-Rad:1652257
Other	CFX Connect Real-Time PCR Detection System	Bio-Rad	Bio-Rad:1855201
Other	Zeiss Axioplan microscope	Zeiss
Other	Nikon Microphot-FX microscope	Nikon
Other	MicroPublisher 3.3 RTV camera	Qimaging