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.
AUTHOR CONTRIBUTIONS
VSD cloned the CrLFY coding sequences and made the RNAi constructs during a sabbatical visit to the University of Oxford; ARP and EHR performed transformations in Ceratopteris richardii and EHR maintained T0 transgenic plants; ARP cloned the CrLFY1 promoter, made GUS reporter constructs, validated transgenic reporter lines, conducted GUS staining, and performed gel blot analysis of CrLFY copy number; SJC and KDHH screened, validated and characterized the RNAi lines; KDHH performed ontogenetic gene expression analysis; VSD performed statistical analyses; SJC conducted in situ localization experiments; JAL performed the phylogenetic analysis; JAL & ARP wrote the first draft of the paper, all authors contributed to the final version.
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 and Bridge Funding Program, University of Washington. We thank Brittany Dean for her contribution to transgenic line screening and validation.
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 and Haughn, 1991; Weigel et al., 1992; Weigel and Nilsson, 1995; 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, 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 complemented by fern and (increasingly) 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 a similar 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), the fern Ceratopteris richardii (hereafter ‘Ceratopteris’) (Himi et al., 2001) and several gymnosperm species (Mellerowicz et al.; Mouradov et al., 1998; Shindo et al., 2001; Carlsbecker et al., 2004; Vázquez-Lobo et al., 2007; Carlsbecker et al., 2013). 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 LFY 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 Files 1-3). The phylogenetic topology inferred within the vascular plants using the entire dataset (Figure 1-figure supplement 1) was consistent with previous analyses (Qiu et al., 2006; Wickett et al., 2014). Within the ferns (64 in total), phylogenetic relationships between LFY sequences indicated that the two gene copies identified in Equisetum arvense, Azolla caroliniana and Ceratopteris each resulted from recent independent duplication events (Figure 1). Gel blot analysis confirmed the presence of no more than two LFY genes in the Ceratopteris genome (Figure 1-figure supplement 2). Given that the topology of the tree excludes the possibility of a gene duplication prior to diversification of the ferns, CrLFY1 and CrLFY2 are equally orthologous to the single copy LFY representatives in other fern species.
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. The sampled developmental stages spanned from imbibed spores prior to germination (Figure 2A), to differentiated male and hermaphrodite gametophytes (Figure 2B-D), through fertilization and formation of the diploid embryo (Figure 2E), to development of the increasingly complex sporophyte body plan (Figure 2F-K). Quantitative RT-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 2L). 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 CrLFY1/2 gene expression that is dependent on developmental stage. Of particular note were significant differences between CrLFY1 and CrLFY2 transcript levels during sporophyte development. Whereas CrLFY2 transcript levels were comparable across sporophyte samples, CrLFY1 transcript levels were much higher in samples that contained the shoot apex (Figure 2F, G) than in those that contained just fronds (Figure 2H-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.
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 LFY 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 development, transgenic lines were generated that expressed the reporter gene B-glucuronidase (GUS) driven by 3.9 kb of the CrLFY1 promoter (CrLFY1pro::GUS) (Supplementary File 5). GUS activity was monitored in individuals from three independent transgenic lines, sampling both before and up to six days after fertilization (Figure 3A-O), using wild-type individuals as negative controls (Figure 3P-T) and individuals from a transgenic line expressing GUS driven by the constitutive 35S promoter (35Spro) (Supplementary File 5) as positive controls (Figure 3U-Y). Notably, no GUS activity was detected in unfertilized archegonia of CrLFY1pro::GUS gametophytes (Figure 3A, F, K) but by two days after fertilization (DAF) GUS activity was detected in most cells of the early sporophyte embryo (Figure 3B, 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 3C, 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 3D, 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.
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 from indeterminate shoot apices (Bierhorst, 1977; White and Turner, 1995). CrLFY1 expression in the shoot apex at the end of embryogenesis (Figure 3E, J, O) and elevated transcript levels in shoot apex-containing sporophyte tissues (Figure 2L) 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 4A-O) and after the transition to reproductive frond formation (Figure 4-figure supplement 1A-L). Wild-type individuals were used as negative controls (Figure 4P-T; Figure 4-figure supplement 1M-P) and 35Spro::GUS individuals as positive controls (Figure 4U-Y; Figure 4-figure supplement 1Q-T). In young sporophytes (20 DAF), GUS activity was primarily localized in shoot apical tissues and newly-emerging frond primordia (Figure 4A, F, K), with very little activity detected in the expanded simple fronds (Figure 4B, G, L). In older vegetative sporophytes (60 DAF), which develop complex dissected fronds (Figure 4C, 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 4D, 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 4E, J, O). In some individuals GUS activity could be detected in frond tissues almost until maturity (Figure 4C). Notably, patterns of CrLFY1pro::GUS expression were the same in the apex and complex fronds of shoots before (60 DAF) (Figure 4C-E, H-J, M-O) and after (~ 115 DAF) the reproductive transition (Figure 4-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 4Z-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.
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) (Supplementary File 6). Phenotypic screening identified 10 lines with similar developmental defects that were associated with reduced CrLFY expression (Table 1).
Wild-type post-embryonic shoot development begins with the production of simple, spade-like fronds from the shoot apex (Figure 5A). In eight transgenic lines, sub-populations of sporophytes developed in which early sporophyte development was perturbed, one line (E8) failing to initiate recognizable embryos (Figure 5B) and the remainder exhibiting premature shoot apex termination, typically after producing several distorted fronds (Figure 5C-H). Sub-populations of phenotypically normal transgenic sporophytes were also identified in some of these lines (Figure 5I-L). The two remaining lines exhibited less severe shoot phenotypes, one (B19) undergoing shoot termination only when wild-type sporophytes produced complex fronds (Figure 5M, N) and the other (D4a) completing sporophyte development but reduced in size to approximately 50% of wild-type (Figure 5O, P). Despite the predicted sequence specificity of CrLFY1-i3 and CrLFY2-i4 (Supplementary File 6), quantitative RT-PCR analysis found that all four RNAi constructs led to suppressed transcript levels of both CrLFY genes (Figure 5Q). The severity of the shoot phenotype was correlated with the level of endogenous CrLFY transcripts detected across all lines (Figure 5Q), with relative levels of both CrLFY1 and CrLFY2 significantly reduced compared to wild-type in all early-terminating sporophytes (p < 0.0001 and p < 0.01, respectively). Notably, phenotypic differences between the two less-severe lines correlated with differences in CrLFY1 transcript levels, which were significantly reduced (p < 0.01) in late-terminating line B19 but not significantly different from wild-type (p > 0.05) in the non-arresting line D4a. CrLFY2 expression was not significantly different from wild-type (p > 0.05) in either the B19 or D4a line, or in the phenotypically normal transgenics. Together these data indicate that wild-type levels of CrLFY2 are sufficient to compensate for some loss of CrLFY1, but at least 10% of wild-type CrLFY1 activity is required to prevent premature termination of the shoot apex. 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.
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 recognized 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 6A). Divisions of the AC go on to form a two-dimensional photosynthetic thallus in both the hermaphrodite (Figure 6B) and male sexes (Figure 6C). 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 6D-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 6D) and in others during AC-driven thallus proliferation (Figure 6E-I). Failure of AC activity was observed in both hermaphrodites (Figure 6E) and males (Figure 6H, I). The phenotypically least severe line exhibited hermaphrodite developmental arrest only after AC activity had been replaced by the notch meristem (Figure 6J). 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 (Figure 6K-N). By contrast CrLFY transcripts were not detected in arrested ZmUbipro::CrLFY1/2-i1 lines (Figure 6O-R) despite confirmed presence of the transgene (Figure 6-figure supplement 1). Although the observed phenotypes could not be ascribed to a specific gene copy, these data support a role for CrLFY 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.
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 2-4). 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 2), 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 5, 6). 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 5). 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 of function 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 6). 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 invoking three sequential changes in gene function during land plant evolution (Figure 7). First, the ancestral LFY function to promote early cell divisions in the embryo was retained as bryophytes and vascular plants diverged, leading to conserved roles in P. patens (Tanahashi et al., 2005) and Ceratopteris (Figure 3). Second, within the vascular plants (preceding divergence of the ferns) this proliferative role expanded to maintain 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 3-5), 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., 2017), 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) 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 (Theißen et al., 2016), the third stage of LFY evolution would 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.; 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.
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, it was the LFY pathway.
MATERIALS AND METHODS
Plant materials and growth conditions
All experimental work was conducted using Ceratopteris richardii strain Hn-n (Hickok et al., 1995). 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. 2017), 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 et al. 1995), Eschscholzia californica EcFLO AAO49794.1 (Busch & 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 & 116935) using Genewise (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.; 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 (Supplementary Files 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 7), 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, 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 Supplementary File 7.
Genomic sequences for CrLFY1 and CrLFY2 ORFs were amplified by PCR from wild-type genomic DNA using primers designed against published transcript sequences (Himi et al., 2001). ORFs of 1551bp and 2108bp 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, Supplementary File 7).
qPCR 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 qPCR primers (Supplementary File 7) were designed spanning intron 1. Amplification specificity of primers was validated via PCR followed by sequencing. qPCR 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 housekeeping genes (Hellemans et al., 2007), CrACTIN1 and CrTATA-BINDING PROTEIN (TBP) (Ganger et al., 2014). The standard deviation of the Ct values of each housekeeping gene was calculated to ensure minimal variation (<3%) in gene expression in wild-type versus transgenic lines.
Relative expression values of CrLFY from qPCR were compared by two-way analysis of variance (ANOVA) for developmental stages or transgenic lines, followed by Sidak’s or Tukey’s multiple comparisons in Prism v. 7.0 (GraphPad Software, Inc., La Jolla, CA). The significance threshold (p) was set at 0.05.
Generation of GUS reporter constructs
The CrLFY1pro::GUS reporter construct (Supplementary File 5) was created by cloning the CrLFY1 promoter (Supplementary File 7) into pART7 as a NotI-XbaI restriction fragment, replacing the existing 35S promoter. A β-Glucuronidase (GUS) coding sequence 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. 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).
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) (Supplementary File 6). Target fragments were amplified from cDNA and cloned into Gateway-compatible entry vector pDONR207. Each sequence was then recombined into the pANDA expression vector via Gateway LR cloning (Invitrogen, Carlsbad, CA).
Generation of transgenic lines
Transformation of all transgenes into wild-type Hn-n Ceratopteris callus was performed as previously described (Plackett et al., 2015). Transgenic lines were assessed in the T1 generation for T-DNA copy number by DNA gel blot analysis and the presence of full-length T-DNA insertions was confirmed through genotyping PCR (Supplementary Files 5 and 6).
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 (1mg/ml X-GlcA, 5uM potassium ferricyanide) were determined empirically. Tissue was cleared with sequential incubations in 70% ethanol until no further decolorization occurred.
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 wild-type 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 521bp 5’UTR and 1113bp ORF; CrLFY2 301bp 5’UTR and 1185bp ORF) (Supplementary File 7). Tissue was fixed in FAA (3.7% formaldehyde, 5% acetic acid; 50 % ethanol) for 1-4 hours 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-published 3.3 RTV camera mounted on a Nikon Microphot-FX microscope. Images were minimally processed for brightness and contrast in Photoshop (CS4).
SUPPLEMENTARY FILES
Supplementary File 1. Additional LFY sequences included in phylogenetic analysis.
Supplementary File 2. Alignment of all LFY amino acid sequences used in phylogenetic analysis.
Supplementary File 3. Alignment of LFY amino acid sequences used in phylogenetic analysis (ferns only).
Supplementary File 4. Statistical comparison of CrLFY transcript levels between different ontogenetic stages
Supplementary File 5. Design and validation of CrLFY1pro::GUS transgenic lines.
Supplementary File 6. Design and validation of CrLFY RNAi transgenic lines.
Supplementary File 7. Gel blot analysis and in situ hybridization probe design.
Supplementary File 8. Summary of published reports of LFY function in a range of angiosperm species.
Footnotes
COMPETING INTERESTS The authors declare that they have no competing interests
AUTHOR CONTRIBUTIONS
VSD cloned the CrLFY coding sequences and made the RNAi constructs during a sabbatical visit to the University of Oxford; ARP and EHR performed transformations in Ceratopteris richardii and EHR maintained T0 transgenic plants; ARP cloned the CrLFY1 promoter, made GUS reporter constructs, validated transgenic reporter lines, conducted GUS staining, and performed gel blot analysis of CrLFY copy number; SJC and KDHH screened, validated and characterized the RNAi lines; KDHH performed ontogenetic gene expression analysis; VSD performed statistical analyses; SJC conducted in situ localization experiments; JAL performed the phylogenetic analysis; JAL & ARP wrote the first draft of the paper, all authors contributed to the final version.