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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
Research Article
Cite this article as: eLife 2018;7:e39625 doi: 10.7554/eLife.39625
8 figures, 2 tables and 9 additional files

Figures

Figure 1 with 2 supplements
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
Figure 1—figure supplement 1
Phylogenetic relationships between LEAFY sequences reflect established relationships within vascular plant lineages.

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

https://doi.org/10.7554/eLife.39625.004
Figure 1—figure supplement 2
The Ceratopteris genome contains only two copies of LFY.

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

https://doi.org/10.7554/eLife.39625.005
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
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
Figure 3—source data 1

CrLFY qRT-PCR ontogenic expression data

https://doi.org/10.7554/eLife.39625.008
Figure 4 with 4 supplements
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
Figure 4—figure supplement 1
Schematic of CrLFY1pro::GUS and 35Spro::GUS constructs.

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

https://doi.org/10.7554/eLife.39625.010
Figure 4—figure supplement 2
DNA gel blot analysis of CrLFY1pro::GUS and 35Spro::GUS transgenic lines.

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

https://doi.org/10.7554/eLife.39625.011
Figure 4—figure supplement 3
PCR analysis of CrLFY1pro::GUS T1 lines identified full-length or near full-length CrLFY1 promoter sequences in T-DNA insertions.

(A) Schematic of the CrLFY1pro::GUS construct marking binding sites of PCR primers used in (B). All positions are given relative to the GUS ATG. Primer sequences are listed in the Key Resources Table. (B) PCR was performed on genomic DNA from the same four individual T1 sporophytes within each line investigated by gel blot analysis (see Figure 4—figure supplement 2). PCR reactions were performed to amplify the native 3.9 kb CrLFY1 promoter as a positive control (row 1) and to amplify T-DNA specific products (rows 2–4) containing the GUS CDS and different lengths of contiguous CrLFY1 promoter sequence (see A). Black arrowheads mark the expected size of the target PCR product in each reaction. PCR analysis identified at least one full-length GUS CDS with ~3.8 kb of CrLFY1 promoter in line AF3 (row 2). Faint PCR products indicate that line AG18 and AE2 probably contain a full-length GUS CDS plus ~3 kb of CrLFY1 promoter sequence (row 3). All lines carry a GUS CDS plus a minimum CrLFY1 promoter length of 875 bp (row 4).

https://doi.org/10.7554/eLife.39625.012
Figure 4—figure supplement 4
PCR analysis of 35Spro::GUS positive control line identified a full-length 35Spro::GUS insertion.

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

https://doi.org/10.7554/eLife.39625.013
Figure 5 with 1 supplement
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
Figure 5—figure supplement 1
CrLFY1pro::GUS expression patterns are similar in Ceratopteris shoots before and after reproductive phase change.

GUS activity detected as blue staining in sporophytes producing fronds with spore-bearing morphology (narrowing and elongation of pinnae) from three independent CrLFY1pro::GUS transgenic reporter lines (A–L); 110–124 DAF), negative wild-type controls (M–P); 113 DAF) and positive 35Spro::GUS controls (Q–T); 110 DAF). Staining patterns were consistent between the three independent CrLFY1pro::GUS transgenic lines (A, E, I), and were similar to those seen at 60 DAF (Figure 4C, (H,M). GUS activity was observed throughout the shoot apex (B, F, J) and in recently-emerged frond primordia (C, G, K). Activity persisted later in frond development, becoming restricted to developing pinnae (D, H, L). GUS staining was lost from fronds prior to maturity (A, E, I). No endogenous GUS activity was detected in wild-type controls (M–P) whereas activity was detected throughout all non-senescent tissues in the 35Spro::GUS line (Q–T).

https://doi.org/10.7554/eLife.39625.015
Figure 6 with 5 supplements
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
Figure 6—source data 1

CrLFY RNAi lines qRT-PCR expression data

https://doi.org/10.7554/eLife.39625.023
Figure 6—figure supplement 1
Positions of CrLFY RNAi target sequences.

Schematic of CrLFY1 and CrLFY2 transcripts, showing recognition sequences used in RNAi constructs (black bars). 5’ and 3’ untranslated regions (UTRs) are marked by grey boxes, coding sequence (CDS) by black boxes, with exons as indicated. Positions are given relative to the translational start codon of each transcript. Four RNAi constructs were generated Two of these (ZmUbipro::CrLFY1/2-i1 and ZmUbipro::CrLFY1/2-i2) targeted both CrLFY1 and CrLFY2 using conserved coding sequence amplified from CrLFY1 (ZmUbipro::CrLFY1/2-i1) or CrLFY2 (ZmUbipro::CrLFY1/2-i2). The two remaining constructs (ZmUbipro::CrLFY1-i3 and ZmUbipro::CrLFY2-i4) incorporate target sequence amplified from the 3’UTR region of CrLFY1 and CrLFY2, respectively. The position of primers used in target sequence amplification are shown. Primer sequences are supplied in the Key Resources table.

https://doi.org/10.7554/eLife.39625.018
Figure 6—figure supplement 2
Generalized schematic of CrLFY RNAi constructs.

Each RNAi construct carries inverted repeats of CrLFY-derived sequence (see Figure 6—figure supplement 1 and Supplementary file 5) to generate a hairpin bridged by a linker sequence derived from the GUS CDS (Miki and Shimamoto, 2004). The positions given are relative to the start of the maize ubiquitin promoter (ZmUbipro) that is driving RNAi expression, and are shown for ZmUbipro::CrLFY1-i3, with the length of the RNAi target sequence varying between the four different constructs (see Figure 6—figure supplement 1). The sites of hybridization against probes for the GUS linker and hygromycin resistance marker (HygR) are shown. The position of a SacI restriction site (used in gel blot analysis, see Figure 6—figure supplement 3) is indicated. No SacI sites are present in any CrLFY target sequence used. The primers used in probe amplification are given in the Key Resources Table.

https://doi.org/10.7554/eLife.39625.019
Figure 6—figure supplement 3
Gel blot analysis of ZmUbipro::CrLFY1-i3 T1 transgenic lines.

ZmUbipro::CrLFY1-i3 lines E8 and G13 were regenerated from two separate bombardments and so are necessarily independent of one another. Genomic DNA was extracted from four T1 sporophytes (arising from the free fertilization of T1 gametophytes) within each line, digested with SpeI and separated on an electrophoresis gel. Gel blot analysis of both constructs was performed with probes hybridizing either to the GUS linker of the RNAi hairpin or to the hygromycin resistance (HygR) CDS (Figure 6—figure supplement 2). For each full-length ZmUbipro::CrLFY1-i3 T-DNA insertion present in the genome a single insert is predicted to result in unlinked hybridization fragments with minimum sizes of ~1 kb (HygR) or ~3.6 kb (GUS linker). Based on the hybridization fragments obtained, line E8 carries two full-length insertions of the CrLFY1 RNAi cassette and line G13 carries two full-length insertions plus two additional partial insertions.

https://doi.org/10.7554/eLife.39625.020
Figure 6—figure supplement 4
Binding site of CrLFY RNAi genotyping PCR primers.

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

https://doi.org/10.7554/eLife.39625.021
Figure 6—figure supplement 5
Genotyping PCR confirms the presence of CrLFY RNAi T-DNA in transgenic lines and the absence of the RNAi hairpin in no hairpin control lines.

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

https://doi.org/10.7554/eLife.39625.022
Figure 7 with 1 supplement
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
Figure 7—figure supplement 1
Gametophytes exhibiting developmental arrest were transgenic.

A. Generalized schematic of CrLFY RNAi T-DNA with the relative position of primers used in genotyping PCR marked. Primer sequences and expected PCR product sizes for each CrLFY RNAi construct are given in Figure 6—figure supplement 4. Genotyping PCR was conducted on DNA extracted from single gametophytes exhibiting developmental arrest at 10 DPS in three ZmUbipro::CrLFY1/2-i1 lines. DNA from all arrested individuals amplified positive bands for the two hairpin arms (pVec8F-PW64R and PW64F-pVec8R) and for the hygromycin resistance marker (HPTF-HPTR).

https://doi.org/10.7554/eLife.39625.025
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

Tables

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
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
assay or kit
iTaq universal SYBR
Green mastermix
Bio-RadBio-Rad:1725120
Commercial
assay or kit
DIG-labelling mixRoche Applied SciencesRoche:11277073910
Commercial
assay or kit
T3 RNA polymeraseRoche Applied SciencesRoche:11031163001
Commercial
assay or kit
T7 RNA polymeraseRoche Applied SciencesRoche:10881767001
Commercial
assay or kit
Anti-DIG antibodyRoche Applied SciencesRoche:11093274910;
RRID:AB_2313639
Commercial
assay or kit
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
Chemical
compound, drug
CTP, [ɑ-32P]Perkin ElmerPerkin Elmer:
BLU008H250UC
Software,
algorithm
IQ-TREENguyen et al. (2015),
PMID:25371430
http://www.iqtree.org/
Software,
algorithm
iTOLLetunic and Bork (2016),
PMID:27095192
https://itol.embl.de/
Software,
algorithm
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,
algorithm
GraphPad PrismGraphPad Software Inc.RRID:SCR_002798https://www.graphpad.com/scientific-software/prism/
Software,
algorithm
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

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3 and 6. Sequences and alignments for phylogenetic analyses are included in Supplementary files 1-3.

Additional files

Supplementary file 1

LFY sequences included in phylogenetic analysis (in addition to Sayou et al., 2014) dataset).

https://doi.org/10.7554/eLife.39625.027
Supplementary file 2

Alignment of all 120 LFY amino acid sequences used in phylogenetic analysis.

https://doi.org/10.7554/eLife.39625.028
Supplementary file 3

Alignment of LFY amino acid sequences used in phylogenetic analysis (ferns only).

https://doi.org/10.7554/eLife.39625.029
Supplementary file 4

Statistical comparison of CrLFY transcript levels between different ontogenetic stages.

https://doi.org/10.7554/eLife.39625.030
Supplementary file 5

Specificity of CrLFY RNAi target sequences.

Alignment (prepared using Clustal Omega) of the full length transcript sequences for CrLFY1 and CrLFY2, with nucleotide identity between the two gene copies denoted by a subtending asterisk. The CDS for each gene is highlighted in bold. The target sequences for each RNAi construct are highlighted in light blue (CrLFY1/2-i1), light green (CrLFY1/2-i2), dark blue (CrLFY1-i3) or dark green (CrLFY2-i4). The CrLFY1/2-i1 and CrLFY1/2-i2 target sequences each have 77% similarity to the opposing gene transcript (BLAST2n, discontiguous megablast for highly similar sequences). The full-length CrLFY1-i3 and CrLFY2-i4 target sequences do not demonstrate significant sequence similarity to the opposing gene transcript or 3’UTR alone (BLAST2n, blastn for somewhat similar sequences) but short regions of similarity within the target sequence might explain the cross-reactivity observed.

https://doi.org/10.7554/eLife.39625.031
Supplementary file 6

Predicted hybridization and specificity of CrLFY in situ hybridization probes.

Alignment (prepared using Clustal Omega) of full length CrLFY1 and CrLFY2 transcript sequences, with nucleotide identity between the two paralogs denoted by a subtending asterisk. The coding sequence (CDS) for each gene copy is highlighted in bold. Predicted sites of hybridization for the two probes are highlighted in blue (CrLFY1) and yellow (CrLFY2) respectively, with PCR primer sites underlined. The in situ probes span the complete CDS and 5’UTR of each gene copy. The CrLFY1 probe sequence shows 79% nucleotide identity to the CrLFY2 transcript (BLAST2n, discontiguous megablast for highly similar sequences). The CrLFY2 probe shows 79% nucleotide identity to the CrLFY1 transcript (BLAST2n, discontiguous megablast for highly similar sequences).

https://doi.org/10.7554/eLife.39625.032
Supplementary file 7

Amplified CrLFY2 genomic fragment (3619 bp), not connected directly to CrLFY2 open reading frame.

Sequence highlighted in green corresponds to published CrLFY2 5’UTR (Himi et al., 2001)

https://doi.org/10.7554/eLife.39625.033
Supplementary file 8

Summary of published reports of LFY function in a range of angiosperm species.

https://doi.org/10.7554/eLife.39625.034
Transparent reporting form
https://doi.org/10.7554/eLife.39625.035

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