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

  1. Andrew RG Plackett
  2. Stephanie J Conway
  3. Kristen D Hewett Hazelton
  4. Ester H Rabbinowitsch
  5. Jane A Langdale  Is a corresponding author
  6. Verónica S Di Stilio  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. University of Washington, United States
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Cite this article as: eLife 2018;7:e39625 doi: 10.7554/eLife.39625

Abstract

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.

https://doi.org/10.7554/eLife.39625.001

eLife digest

The first plants colonized land around 500 million years ago. These plants had simple shoots with no branches, similar to the mosses that live today. Later on, some plants evolved more complex structures including branched shoots and flowers (collectively known as the “flowering plants”). Ferns are a group of plants that evolved midway between the mosses and flowering plants and have branched shoots but no flowers.

The gradual transition from simple to more complex plant structures required changes to the way in which cells divide and grow within plant shoots. Whereas animals produce new cells throughout their body, most plant cells divide in areas known as meristems. All plants grow from embryos, which contain meristems that will form the roots and shoots of the mature plant. A gene called LEAFY is required for cells in moss embryos to divide. However, in flowering plants LEAFY does not carry out this role, instead it is only required to make the meristems that produce flowers.

How did LEAFY transition from a general role in embryos to a more specialized role in making flowers? To address this question, Plackett, Conway et al. studied the two LEAFY genes in a fern called Ceratopteris richardii. The experiments showed that at least one of these LEAFY genes was active in the meristems of fern shoots throughout the lifespan of the plant. The shoots of ferns with less active LEAFY genes could not form the leaves seen in normal C. richardii plants. This suggests that as land plants evolved, the role of LEAFY changed from forming embryos to forming complex shoot structures.

Most of our major crops are flowering plants. By understanding how the role of LEAFY has changed over the evolution of land plants, it might be possible to manipulate LEAFY genes in crop plants to alter shoot structures to better suit specific environments.

https://doi.org/10.7554/eLife.39625.002

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 13). 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 with 2 supplements see all
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.

https://doi.org/10.7554/eLife.39625.003

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).

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.

https://doi.org/10.7554/eLife.39625.006

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.

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.

https://doi.org/10.7554/eLife.39625.007

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 (CrLFY1pro::GUS). 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 14. 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 (35Spro) as positive controls (Figure 4U–Y). Notably, no GUS activity was detected in unfertilized archegonia of CrLFY1pro::GUS 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 with 4 supplements see all
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 CrLFY1pro::GUS transgenic reporter lines (A–O), a representative negative wild-type control line (P–T) and a representative positive 35Spro::GUS 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 CrLFY1pro::GUS 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 35Spro::GUS 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.

https://doi.org/10.7554/eLife.39625.009

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). CrLFY1pro::GUS 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 CrLFY1pro::GUS 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 35Spro::GUS 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 CrLFY1pro::GUS 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 with 1 supplement see all
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 CrLFY1pro::GUS transgenic reporter lines (A–O), negative wild-type controls (P–T) and positive 35Spro::GUS 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 CrLFY1pro::GUS 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 CrLFY1pro::GUS 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 CrLFY1pro::GUS fronds (AB–AD). Scale bars = 10 mm (Z, AA), 500 μm (AB–AD).

https://doi.org/10.7554/eLife.39625.014

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 (ZmUbipro). Construct maps plus DNA blot and PCR validation of transgenic lines are shown in Figure 6—figure supplements 15. 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).

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 ZmUbipro::CrLFY1/2-i1 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.

https://doi.org/10.7554/eLife.39625.016
RNAi transgeneLineTransfor-mation replicateGametophyte phaseSporophyte phase
 Spore germin-ationAC-based growthNotch meristem-based growth% arrestedEmbryoShoot apex initiatedSimple frondComplex frond% arrested
ZmUBIpro::CrLFY1/2-i1B161+--99.86++--<5%
ZmUBIpro::CrLFY1/2-i1B191+--50.00+++-<5%
ZmUBIpro::CrLFY1/2-i1D132+--99.80++--<5%
ZmUBIpro::CrLFY1/2-i1D23+++0.00++--<5%
ZmUBIpro::CrLFY1/2-i1D43+++0.00++++<5%
ZmUBIpro::CrLFY1/2-i2F94+--0.00++--<5%
ZmUBIpro::CrLFY1/2-i2F145---100.00----0
ZmUBIpro::CrLFY1-i3E86++/-+/-100.00----0
ZmUBIpro::CrLFY1-i3G137+++0.00++--<5%
ZmUBIpro::CrLFY2-i4C38+++0.00++--<5%
NHC-1 (control)D209+++0.00++++0
NHC-2 (control)F310+++0.00++++0
NHC-3 (control)F410+++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 with 5 supplements see all
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 (ZmUbipro::CrLFY1-i3) (B, C), CrLFY2 (ZmUbipro::CrLFY1-i4) (D) and both CrLFY1 and CrLFY2 (ZmUbipro::CrLFY1/2-i1 and ZmUbipro::CrLFY1/2-i2) (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 ZmUbipro::CrLFY1/2-i1 (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).

https://doi.org/10.7554/eLife.39625.017

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 ZmUbipro::CrLFY1-i3, ZmUbipro::CrLFY1/2-i1 or ZmUbipro::CrLFY1/2-i2) 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 ZmUbipro::CrLFY1/2-i1 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 with 1 supplement see all
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 ZmUbipro::CrLFY1/2-i1 lines CrLFY transcripts could not be detected (O–R), and transgene presence was confirmed (Figure 7—figure supplement 1). Scale bars = 100 μm.

https://doi.org/10.7554/eLife.39625.024

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 35). 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 46), 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.

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).

https://doi.org/10.7554/eLife.39625.026

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

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional 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-i1This paperC. richardii transgenic line;
RNAi knockdown of CrLFY1
and CrLFY2 expression.
Genetic reagent
(C. richardii)
CrLFY1/2-i2This paperC. richardii transgenic line;
RNAi knockdown of CrLFY1
and CrLFY2 expression.
Genetic reagent
(C. richardii)
CrLFY1-i3This paperC. richardii transgenic line;
RNAi knockdown of CrLFY1
expression.
Genetic reagent
(C. richardii)
CrLFY2-i4This paperC. richardii transgenic line;
RNAi knockdown of CrLFY2
expression.
Genetic reagent
(C. richardii)
CrLFY1pro::GUSThis paperC. richardii transgenic line;
CrLFY1pro::GUS reporter.
Genetic reagent
(C. richardii)
35Spro::GUSThis paperC. richardii transgenic line;
35Spro::GUS reporter.
Recombinant
DNA reagent
CrLFY1proThis paperNCBI:MH841970CrLFY1 5' genomic fragment;
Figure 1—figure supplement 2
Recombinant
DNA reagent
CrLFY2pro fragment 1This paperNCBI:MH841971CrLFY2 5' genomic fragment;
Figure 1—figure supplement 2
Recombinant
DNA reagent
CrLFY2pro fragment 2This paperCrLFY2 5' genomic fragment;
Figure 1—figure supplement 2;
Supplementary file 7
Recombinant
DNA reagent
GUSUlmasov, 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)InvitrogenThermo Scientific:
K457502
Recombinant
DNA reagent
pDONR207 (Gateway vector)Invitrogen
Recombinant
DNA reagent
pBOMBER (Binary vector)Plackett et al. (2015),
PMID:26146510
NCBI:MH841969Modified 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-pANDAThis paperRNAi construct targeting CrLFY1
and CrLFY2;
Figure 6—figure supplement 1;
Figure 6—figure supplement 2
Recombinant
DNA reagent
ZmUbipro::CrLFY1/2-i2-pANDAThis paperRNAi construct targeting
CrLFY1 and CrLFY2;
Figure 6—figure supplement 1;
Figure 6—figure supplement 2
Recombinant
DNA reagent
ZmUbipro::CrLFY1-i3-pANDAThis paperRNAi construct targeting CrLFY1;
Figure 6—figure supplement 1;
Figure 6—figure supplement 2
Recombinant
DNA reagent
ZmUbipro::CrLFY2-i4-pANDAThis paperRNAi construct targeting CrLFY2;
Figure 6—figure supplement 1;
Figure 6—figure supplement 2
Recombinant
DNA reagent
CrLFY1pro::GUS-pBOMBERThis paperGUS reporter construct, CrLFY1;
Figure 4—figure supplement 1
Recombinant
DNA reagent
35Spro::GUS-pBOMBERThis paperGUS reporter construct, 35S
control; Figure 4—figure supplement 1
Recombinant
DNA reagent
CrLFY1 in situ probe (antisense)This paperIn situ hybridisation probe;
Supplementary file 6
Recombinant
DNA reagent
CrLFY1 in situ probe (sense)This paperIn situ hybridisation probe;
Supplementary file 6
Recombinant
DNA reagent
CrLFY2 in situ probe (antisense)This paperIn situ hybridisation probe;
Supplementary file 6
Recombinant
DNA reagent
CrLFY2 in situ probe (sense)This paperIn situ hybridisation probe;
Supplementary file 6
Recombinant
DNA reagent
32P-CrLFY1 probe 1This paperDNA gel blot probe for
CrLFY1;
Figure 1—figure supplement 2
Recombinant
DNA reagent
32P-CrLFY1 probe 2This paperDNA gel blot probe for CrLFY1;
Figure 1—figure supplement 2
Recombinant
DNA reagent
32P-CrLFY2 probe 1This paperDNA gel blot probe for CrLFY2;
Figure 1—figure supplement 2
Recombinant
DNA reagent
32P-CrLFY2 probe 1This paperDNA gel blot probe for CrLFY2;
Figure 1—figure supplement 2
Recombinant
DNA reagent
32P-HygR probePlackett et al. (2014),
PMID:24623851
DNA gel blot probe, T-DNA
specific; Figure 4—figure supplement 1
Recombinant
DNA reagent
32P-GUS probePlackett et al. (2014),
PMID:24623851
DNA gel blot probe, T-DNA specific;
Figure 4—figure supplement 1
Recombinant
DNA reagent
32P-GUSlinker probeThis paperDNA gel blot probe, T-DNA specific;
Figure 6—figure supplement 2
Sequence-based
reagent
CrLFY1ampFThis paperORF amplification,
CrLFY1: 5'-ATGGATGTCTCT
TTATTGCCAC-3'
Sequence-based
reagent
CrLFY1ampRThis paperORF amplification,
CrLFY1: 5'-TCAATCATAGATGC
AGCTATCACTG-3'
Sequence-based
reagent
CrLFY1ampFThis paperORF amplification,
CrLFY2: 5'-ATGTTCCGATGG
GAACAAAG-3'
Sequence-based
reagent
CrLFY1ampRThis paperORF amplification,
CrLFY2: 5'-TTATTCATAGCT
GCAGCTGTC-3'
Sequence-based
reagent
CrLFY1invFThis paperInverse PCR,
CrLFY1: 5'-CTATGGAGTAC
GAAGCACCAC-3'
Sequence-based
reagent
CrLFY1invF2This paperInverse PCR,
CrLFY1: 5'-CGATCATTTCTT
GTACTGCTCTC-3'
Sequence-based
reagent
CrLFY1invF3This paperInverse PCR, CrLFY1
: 5'-CAGTGCATGACCTTCGATATTG-3'
Sequence-based
reagent
CrLFY1invRThis paperInverse PCR, CrLFY1:
5'-CAGTTGTTTCGGATCTGCAG-3'
Sequence-based
reagent
CrLFY1invR2This paperInverse PCR, CrLFY1:
5'-CTCCGCTTTTCATTTGAGAACG-3'
Sequence-based
reagent
CrLFY1invR3This paperInverse PCR, CrLFY1:
5'-CAAGAACCGCTGGAGTAAAC-3'
Sequence-based
reagent
CrLFY2invFThis paperInverse PCR, CrLFY2:
5'-CTATGGTGTACGGAGCACTAC-3'
Sequence-based
reagent
CrLFY2invF2This paperInverse PCR,
CrLFY2: 5'-CGTATCCAAAACAGC
TTAAACTCC-3'
Sequence-based
reagent
CrLFY2invF3This paperInverse PCR, CrLFY2:
5'-CACTAAAGGTGCTGCTATCAAC-3'
Sequence-based
reagent
CrLFY2invF4This paperInverse PCR, CrLFY2:
5'-CATTGTGCTGACCTTGTGAAG-3'
Sequence-based
reagent
CrLFY2invF5This paperInverse PCR,
CrLFY2: 5'-CGCAAAGGTTGGAA
AAGAGAAC-3'
Sequence-based
reagent
CrLFY2invF6This paperInverse PCR, CrLFY2:
5'-CGACAACGGATCATAACCATC-3'
Sequence-based
reagent
CrLFY2 invF7This paperInverse PCR,
CrLFY2: 5'-CAATAGTAGATT
CTCCCTCCTTTAC-3'
Sequence-based
reagent
CrLFY2invF8This paperInverse PCR,
CrLFY2: 5'-GCTCTTTAATTT
GAATCACGTGTG-3'
Sequence-based
reagent
CrLFY2invF9This paperInverse PCR,
CrLFY2: 5'-GAACAATGTGCA
TGCGACTC-3'
Sequence-based
reagent
CrLFY2invF10This paperInverse PCR,
CrLFY2: 5'-CATGTTCCGAT
GGGAACAAAG-3'
Sequence-based
reagent
CrLFY2invF11This paperInverse PCR,
CrLFY2: 5'-CATAGGGAACT
CTGTAATGATGC-3'
Sequence-based
reagent
CrLFY2invF12This paperInverse PCR,
CrLFY2: 5'-GTTTCCAG
ATACTGCTGCTC-3'
Sequence-based
reagent
CrLFY2invF13This paperInverse PCR,
CrLFY2: 5'-CATAGATGA
TGCCAGTATACTCC-3'
Sequence-based
reagent
CrLFY2invF14This paperInverse PCR,
CrLFY2: 5'-GCTCACTAT
CCACAATTCATACAC-3'
Sequence-based
reagent
CrLFY2invF15This paperInverse PCR,
CrLFY2: 5'-GTTCGTATCT
GATACTTGTTTCGTG-3'
Sequence-based
reagent
CrLFY2invF16This paperInverse PCR,
CrLFY2: 5'-CTTACTCCA
CGAATGCATGC-3'
Sequence-based
reagent
CrLFY2invRThis paperInverse PCR,
CrLFY2: 5'-CAGTTGTCAC
AGAGGTAGCAG-3'
Sequence-based
reagent
CrLFY2invR2This paperInverse PCR,
CrLFY2: 5'-CCTTACGATG
TATTACCCTTTGTTC-3'
Sequence-based
reagent
CrLFY2invR3This paperInverse PCR,
CrLFY2: 5'-CAGTGACTA
GGATGTCTGATACAG-3'
Sequence-based
reagent
CrLFY2invR4This paperInverse PCR,
CrLFY2: 5'-GAAGGAGCT
GAAAATGCAACTC-3'
Sequence-based
reagent
CrLFY2invR5This paperInverse PCR,
CrLFY2: 5'-CCTGCCTCC
TATGAAAACAC-3'
Sequence-based
reagent
CrLFY2invR6This paperInverse PCR,
CrLFY2: 5'-CCTGTAAAGG
AGGGAGAATCTAC-3'
Sequence-based
reagent
CrLFY2invR7This paperInverse PCR,
CrLFY2: 5'-GCACTCCAAC
GATGATGATAC-3'
Sequence-based
reagent
CrLFY2invR8This paperInverse PCR,
CrLFY2: 5'-GCTGTACTA
AGGCATCAATTCAG-3'
Sequence-based
reagent
CrLFY2invR9This paperInverse PCR,
CrLFY2: 5'-CATCTATGATA
GCACAACATCACTC-3'
Sequence-based
reagent
CrLFY2invR10This paperInverse PCR,
CrLFY2: 5'-CACAACATC
ACTCAGGACTC-3'
Sequence-based
reagent
CrLFY2invR11This paperInverse PCR,
CrLFY2: 5'-CTGCCTCCTA
TGAAAACACAAG-3'
Sequence-based
reagent
CrLFY2invR12This paperInverse PCR,
CrLFY2: 5'-CTAGTCTTTG
ATGAGGTTTCATGTC-3'
Sequence-based
reagent
CrLFY2invR13This paperInverse PCR,
CrLFY2: 5'-CATGCAAGA
AGCATGCAATTC-3'
Sequence-based
reagent
CrLFY2invR14This paperInverse PCR,
CrLFY2: 5'-GTGTCTCCA
GTAAGTATGAAACAAG-3'
Sequence-based
reagent
CrLFY2invR15This paperInverse PCR,
CrLFY2: 5'-CATGAGGCC
GTCAGACTTAC-3'
Sequence-based
reagent
CrLFY2invR16This paperInverse PCR,
CrLFY2: 5'-CGTAACAGA
CGAGCTCGATATAATAG-3'
Sequence-based
reagent
CrLFY2invR17This paperInverse PCR,
CrLFY2: 5'-CTCTTTGCTCA
TATAGCTTCAAGC-3'
Sequence-based
reagent
CrLFY1 + 2 (1)-RNAi-FThis paperT-DNA cloning,
CrLFY1/2-i1: 5'-ATGGGT
TTCACTGTGAATAC-3'
Sequence-based
reagent
CrLFY1 + 2 (1)-RNAi-RThis paperT-DNA cloning,
CrLFY1/2-i1: 5'-TCTCCTC
TTTGTTCCCTTGTG-3'
Sequence-based
reagent
CrLFY1 + 2 (2)-RNAi-FThis paperT-DNA cloning,
CrLFY1/2-i2: 5'-ATGGG
TTTCACTGTTAGTAC-3'
Sequence-based
reagent
CrLFY1 + 2 (2)-RNAi-RThis paperT-DNA cloning,
CrLFY1/2-i2: 5'-TCTCCT
CTTTGTTCCCTGGTG-3'
Sequence-based
reagent
CrLFY1-RNAi-FThis paperT-DNA cloning,
CrLFY1-i3: 5'-CCTTTTCT
TGCTAATGATGGC-3'
Sequence-based
reagent
CrLFY1-RNAi-RThis paperT-DNA cloning,
CrLFY1-i3: 5'-CAAACAAA
CTTGAAAATGATAC-3'
Sequence-based
reagent
CrLFY2-RNAi-FThis paperT-DNA cloning,
CrLFY2-i4: 5'-GCCATTG
CTAGCAAGGTTAT-3'
Sequence-based
reagent
CrLFY2-RNAi-RThis paperT-DNA cloning,
CrLFY2-i4: 5'-CACTGCT
TTGAAACTAAAAC-3'
Sequence-based
reagent
pCrLFY1amp-NotFThis paperT-DNA cloning,
CrLFY1pro: 5'-CAGCGGCCGCTTAGATGG
CTTGAGATGCTAC-3'
Sequence-based
reagent
pCrLFY1amp-XbaRThis paperT-DNA cloning,
CrLFY1pro: 5'-CATCTAGAG
GAGGCACTTCTTTACGTG-3'
Sequence-based
reagent
GUSamp-XbaFThis paperT-DNA cloning,
GUS CDS: 5'-CATCTAGAC
AATGGTAAGCTTAGCGGG-3'
Sequence-based
reagent
GUSamp-XbaRThis paperT-DNA cloning,
GUS CDS: 5'-CCATCTAGA
TTCATTGTTTGCCTCCCTG-3'
Sequence-based
reagent
qCrLFY1_F2This paperqRT-PCR,
CrLFY1: 5'-GTCCGCT
ATTCGTGCAGAGA-3'
Sequence-based
reagent
qCrLFY1_R2This paperqRT-PCR, CrLFY1
: 5'-AATTCAAGGGGG
CATTGGGT-3'
Sequence-based
reagent
qCrLFY2_F3This paperqRT-PCR, CrLFY2:
5'-GCAGTGACAATGAAGGACGC-3'
Sequence-based
reagent
qCrLFY2_R3This paperqRT-PCR, CrLFY2:
5'-AGAATCGTGCACACTGCTCA-3'
Sequence-based
reagent
qCrTBPb_FGanger et al. (2015),
DOI:10.1139/cjb-2014–0202
qRT-PCR, CrTBP:
5'-ATGAGCCAGAGCTTTTCCCC-3'
Sequence-based
reagent
qCrTBPb_RGanger et al. (2015),
DOI:10.1139/cjb-2014–0202
qRT-PCR, CrTBP:
5'-TTCGTCTCTGACCTTTGCCC-3'
Sequence-based
reagent
qCrACT1_FGanger et al. (2015),
DOI:10.1139/cjb-2014–0202
qRT-PCR,
CrActin1: 5'-GAGAGAGGCTA
CTCTTTCACAACC-3'
Sequence-based
reagent
qCrACT1_RGanger et al. (2015),
DOI:10.1139/cjb-2014–0202
qRT-PCR,
CrActin1: 5'-AGGAAGTTCGTA
ACTCTTCTCCAA-3'
Sequence-based
reagent
CrLFY1_ISH_FThis paperIn situ probes,
CrLFY1: 5'-GAGGCATACA
CACACGCAGT-3'
Sequence-based
reagent
CrLFY1_ISH_RThis paperIn situ probes,
CrLFY1: 5'-TCAATCATAGAT
GCAGCTATCACTG-3
Sequence-based
reagent
CrLFY2_ISH_FThis paperIn situ probes,
CrLFY2: 5'-GGCTGGTTGTTA
CGGATAGC-3'
Sequence-based
reagent
CrLFY2_ISH_RThis paperIn situ probes,
CrLFY2: 5'-TTATTCATAG
CTGCAGCTGTCACTG-3'
Sequence-based
reagent
CrLFY1_Probe1FThis paperCopy number analysis,
CrLFY1 probe 1: 5'-CAGG
CACAAGGGAACAAAG-3'
Sequence-based
reagent
CrLFY1_Probe1RThis paperCopy number analysis,
CrLFY1 probe 1: 5'-CA
TAGATGCAGCTATCACTGTC-3'
Sequence-based
reagent
CrLFY1_Probe2FThis paperCopy number analysis,
CrLFY1 probe 2:
5'-CACTTGAAGGTAAGCT
TTATTGTAAGG-3'
Sequence-based
reagent
CrLFY1_Probe2RThis paperCopy number analysis,
CrLFY1 probe 2: 5'-CAATA
TTTCCGACTATACATTGAGGC-3'
Sequence-based
reagent
CrLFY2_Probe1FThis paperCopy number analysis,
CrLFY2 probe 1: 5'-CAGGCA
CCAGGGAACAAAG-3'
Sequence-based
reagent
CrLFY2_Probe1RThis paperCopy number analysis,
CrLFY2 probe 1: 5'-CATAGC
TGCAGCTGGTCACTGTC-3'
Sequence-based
reagent
CrLFY2_Probe2FThis paperCopy number analysis,
CrLFY2 probe 2: 5'-CTGTAG
AAGGTAAGATTCTGCTC-3'
Sequence-based
reagent
CrLFY2_Probe2RThis paperCopy number analysis,
CrLFY2 probe 2: 5'-GCTT
ATGGTACAGAATAAGTAGAGG-3'
Sequence-based
reagent
HygF2Plackett et al. (2014),
PMID:24623851
T-DNA gel blot
probe, HygR: 5'-CTTCTACA
CAGCCATCGGTC-3'
Sequence-based
reagent
HygRPlackett et al. (2014),
PMID:24623851
T-DNA gel blot
probe, HygR: 5'-CCGATGGT
TTCTACAAAGATCG-3'
Sequence-based
reagent
GH3seqF3Plackett et al. (2014),
PMID:24623851
T-DNA gel blot
probe, GUS: 5'-CTTCGCT
GTACAGTTCTTTCG-3'
Sequence-based
reagent
GH3seqR4Plackett et al. (2014),
PMID:24623851
T-DNA gel blot
probe, GUS: 5'-CACTCATT
ACGGCAAAGTGTG-3'
Sequence-based
reagent
GUSlinkerseqFThis paperT-DNA gel blot
probe, RNAi: 5'-CTGATT
AACCACAAACCGTTCTAC-3'
Sequence-based
reagent
GUSlinkerseqRThis paperT-DNA gel blot
probe, RNAi: 5'-CTGATA
CTCTTCACTCCACATG-3'
Sequence-based
reagent
HPT-FMiki and Shimamoto (2004),
PMID:15111724
RNAi genotyping,
HygR: 5'-GAGCCTGACCTA
TTGCATCTCC-3'
Sequence-based
reagent
HPT-RMiki and Shimamoto (2004),
PMID:15111724
RNAi genotyping,
HygR: 5'-GGCCTCCAG
AAGAAGATGTTGG-3'
Sequence-based
reagent
pVec8FMiki and Shimamoto (2004),
PMID:15111724
RNAi genotyping,
RNAi hairpin: 5'-TTTAGC
CCTGCCTTCATACG-3'
Sequence-based
reagent
pVec8RMiki and Shimamoto (2004),
PMID:15111724
RNAi genotyping,
RNAi hairpin: 5'-ATTGC
CAAATGTTTGAACGA-3'
Sequence-based
reagent
PW64FThis paperRNAi genotyping,
RNAi hairpin: 5'-CATGAA
GATGCGGACTTACG-3'
Sequence-based
reagent
PW64RThis paperRNAi genotyping,
RNAi hairpin: 5'-ATCCAC
GCCGTATTCGG-3'
Sequence-based
reagent
pCrLFY1genoF1This paperCrLFY1pro::GUS
genotyping: 5'-CTTAGA
TGGCTTGAGATGCTAC-3'
Sequence-based
reagent
pCrLFY1genoF2This paperCrLFY1pro::GUS
genotyping: 5'-CTCTCT
TCTTGCTTGTGTTGTG-3'
Sequence-based
reagent
pCrLFY1genoF3This paperCrLFY1pro::GUS genotyping:
5'-CAACTGGCAACAGGTGATG-3'
Sequence-based
reagent
pCrLFY1genoF4This paperCrLFY1pro::GUS genotyping:
5'-CAGTCTTAGTTCAACTGCATTCG-3'
Sequence-based
reagent
pCrLFY1genoRThis paperCrLFY1pro::GUS genotyping:
5'-AGGAGGCACTTCTTTACGTG-3'
Sequence-based
reagent
GUSgenoRThis paperCrLFY1pro::GUS + 35Spro::GUS
genotyping: 5'-CATTGTTTG
CCTCCCTGC-3'
Sequence-based
reagent
35SgenoFThis paper35Spro::GUS genotyping:
5'-CTGAGCTTAACAGCACAGTTG-3'
Sequence-based
reagent
OCS3’genoRThis paper35Spro::GUS genotyping:
5'-CATCACTAGTAAGCTAGCTTGC-3'
Commercial
assay or kit
Phusion high-fidelity
polymerase
Thermo ScientificThermo Scientific:
F530S
Commercial
assay or kit
Gateway LR clonase
II enzyme mix
InvitrogenThermo Scientific:
11791100
Commercial
assay or kit
QIAGEN Plasmid
Maxi Kit
QIAGENQIAGEN:12163
Commercial
assay or kit
Whatman Nytran
nylon blotting membrane
GE HealthcareGE Healthcare:
10416294
Commercial
assay or kit
Random Primers
DNA Labelling kit
InvitrogenThermo Scientific:
18187013
Commercial
assay or kit
Carestream Kodak
autoradiography GBX
developer and fixer
Sigma-AldrichSigma-Aldrich:
Z354147
Commercial
assay or kit
Carestream Kodak
Biomax XAR film
Sigma-AldrichSigma-Aldrich:F5763
Commercial
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iTaq universal SYBR
Green mastermix
Bio-RadBio-Rad:1725120
Commercial
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DIG-labelling mixRoche Applied SciencesRoche:11277073910
Commercial
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T3 RNA polymeraseRoche Applied SciencesRoche:11031163001
Commercial
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T7 RNA polymeraseRoche Applied SciencesRoche:10881767001
Commercial
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Anti-DIG antibodyRoche Applied SciencesRoche:11093274910;
RRID:AB_2313639
Commercial
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NBT/BCIP stock solutionRoche Applied SciencesRoche:11681451001
Chemical
compound, drug
Potassium ferricyanide
(K₃Fe(CN)₆)
Sigma-AldrichSigma:P8131
Chemical
compound, drug
X-GlcA (CHA salt)Melford ScientificMelford:MB1021
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CTP, [ɑ-32P]Perkin ElmerPerkin Elmer:
BLU008H250UC
Software,
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IQ-TREENguyen et al. (2015),
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Software,
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iTOLLetunic and Bork (2016),
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https://itol.embl.de/
Software,
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ClustalWLi et al. (2015),
PMID:25845596
RRID:SCR_002909https://www.ebi.ac.uk/Tools/msa/clustalw2/
Software,
algorithm
TBLASTXAltschul et al. (1990),
PMID:2231712
RRID:SCR_011823https://blast.ncbi.nlm.nih.gov/Blast.cgi
Software,
algorithm
GeneWiseBirney et al. (2004),
PMID:15123596
RRID:SCR_015054https://www.ebi.ac.uk/Tools/psa/genewise
Software,
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GraphPad PrismGraphPad Software Inc.RRID:SCR_002798https://www.graphpad.com/scientific-software/prism/
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Adobe Photoshop CS4AdobeRRID:SCR_014199
OtherBiolistic PDS-1000/He
Particle Delivery System
Bio-RadBio-Rad:1652257
OtherCFX Connect Real-Time
PCR Detection System
Bio-RadBio-Rad:1855201
OtherZeiss Axioplan microscopeZeiss
OtherNikon Microphot-FX
microscope
Nikon
OtherMicroPublisher
3.3 RTV camera
Qimaging

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 (Figure 1—figure supplement 1).

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 CrLFY1pro::GUS 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 35Spro::GUS positive control (Figure 4—figure supplement 4). The resulting CrLFY1pro::GUS::ocs and 35Spro::GUS::ocs 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.

GUS staining

GUS activity analysis in CrLFY1pro::GUS 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).

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Decision letter

  1. Christian S Hardtke
    Senior Editor; University of Lausanne, Switzerland
  2. Sheila McCormick
    Reviewing Editor; University of California-Berkeley, United States

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 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.

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.

https://doi.org/10.7554/eLife.39625.037

Author response

[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.

https://doi.org/10.7554/eLife.39625.038

Article and author information

Author details

  1. Andrew RG Plackett

    Department of Plant Sciences, University of Oxford, Oxford, United Kingdom
    Present address
    Department of Plant Sciences, University of Cambridge, Cambridge, United Kigndom
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Cloned open reading frames and upstream promoter fragments of CrLFY1 and CrLFY2, Made the CrLFY1pro::GUS reporter constructs, Generated and validated transgenic reporter lines, Conducted GUS staining, Performed gel blot analysis of CrLFY copy number
    Contributed equally with
    Stephanie J Conway
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2321-7849
  2. Stephanie J Conway

    Department of Biology, University of Washington, Seattle, United States
    Present address
    Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, United States
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Screened, validated and characterized RNAi lines, Carried out in situ hybridizations
    Contributed equally with
    Andrew RG Plackett
    Competing interests
    No competing interests declared
  3. Kristen D Hewett Hazelton

    Department of Biology, University of Washington, Seattle, United States
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Screened, validated and characterized RNAi lines, Performed ontogenetic gene expression analysis
    Competing interests
    No competing interests declared
  4. Ester H Rabbinowitsch

    Department of Plant Sciences, University of Oxford, Oxford, United Kingdom
    Contribution
    Investigation, Generated and maintained T0 transgenic lines
    Competing interests
    No competing interests declared
  5. Jane A Langdale

    Department of Plant Sciences, University of Oxford, Oxford, United Kingdom
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Carried out the phylogenetic analyses
    For correspondence
    jane.langdale@plants.ox.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7648-3924
  6. Verónica S Di Stilio

    Department of Biology, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Cloned the CrLFY coding sequences and made the RNAi constructs during a sabbatical visit to the University of Oxford, Carried out the statistical analyses
    For correspondence
    distilio@uw.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6921-3018

Funding

Horizon 2020 Framework Programme (ERC AdG EDIP)

  • Jane A Langdale

Gatsby Charitable Foundation

  • Jane A Langdale

National Science Foundation (EDEN IOS 0955517)

  • Verónica S Di Stilio

University of Washington (Royalty Research Fund, A96605)

  • Verónica S Di Stilio

University of Washington (Bridge Funding Program 652315)

  • Verónica S Di Stilio

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Work in JAL’s lab was funded by an ERC Advanced Investigator Grant (EDIP) and by the Gatsby Charitable Foundation. VSD’s visit to JAL’s lab was funded in part by NSF/EDEN IOS 0955517. Work in VSD’s lab was funded by the Royalty Research Fund (A96605) and Bridge Funding Program (652315), University of Washington. We thank Brittany Dean, Henry Blazina and Jonathan Yee for their contribution to transgenic line screening and validation. We are grateful to Peng Wang for the primers PW64F and PW64R.

Senior Editor

  1. Christian S Hardtke, University of Lausanne, Switzerland

Reviewing Editor

  1. Sheila McCormick, University of California-Berkeley, United States

Publication history

  1. Received: June 27, 2018
  2. Accepted: September 22, 2018
  3. Version of Record published: October 24, 2018 (version 1)

Copyright

© 2018, Plackett et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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