Conserved Regulatory Mechanism Controls the Development of Cells

Conserved regulatory mechanism controls the PNAS PLUS development of cells with rooting functions in land plants

Thomas Ho Yuen Tam, Bruno Catarino, and Liam Dolan1

Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom

Edited by Philip N. Benfey, Duke University, Durham, NC, and approved June 11, 2015 (received for review August 22, 2014) Land plants develop filamentous cells—root hairs, rhizoids, and the moss Physcomitrella patens (8–12). To test whether the con- caulonemata—at the interface with the soil. Members of the servation of RSL function is unique or indicative of a more gen- group XI basic helix–loop–helix (bHLH) transcription factors en- eral, conserved regulatory mechanism, we determined whether coded by LOTUS JAPONICUS ROOTHAIRLESS1-LIKE (LRL) genes other components of the gene-regulatory network controlling an- positively regulate the development of root hairs in the angio- giosperm root-hair development are conserved among land plants. sperms Lotus japonicus, Arabidopsis thaliana, and rice (Oryza sat- Group XI bHLH transcription factors encoded by LOTUS iva). Here we show that auxin promotes rhizoid and caulonema JAPONICUS ROOTHAIRLESS1-LIKE (LRL) genes are also development by positively regulating the expression of PpLRL1 key regulators of root-hair development in angiosperms. LRL and PpLRL2, the two LRL genes in the Physcomitrella patens ge- genes encode several proteins that regulate root-hair devel- nome. Although the group VIII bHLH proteins, AtROOT HAIR opment in diverse angiosperms, including ROOTHAIRLESS1 DEFECTIVE6 and AtROOT HAIR DEFECTIVE SIX-LIKE1, promote root- (LjRHL1) in Lotus japonicus, AtLRL1, AtLRL2, AtLRL3 in hair development by positively regulating the expression of AtLRL3 A. thaliana (13, 14), and ROOTHAIRLESS1 (OsRHL1) in Oryza in A. thaliana, LRL genes promote rhizoid development indepen- sativa (15). Other LRL genes include OsPTF1, involved in dently of PpROOT HAIR DEFECTIVE SIX-LIKE1 and PpROOT HAIR phosphate starvation tolerance in rice (16), and AtUNE12, in- DEFECITVE SIX-LIKE2 (PpRSL1 and PpRSL2) gene function in volved in female gametophyte development in Arabidopsis PLANT BIOLOGY P. patens. Together, these data demonstrate that both LRL and (17). Here we define the function of LRL genes in the moss RSL genes are components of an ancient auxin-regulated gene P. patens and conclude that, together, LRL and RSL genes form network that controls the development of tip-growing cells with the core of an ancient network that operated in the common rooting functions among most extant land plants. Although this net- ancestor of the mosses and angiosperms that existed in the work has diverged in the moss and the angiosperm lineages, our data Silurian Period 415–435 million years ago. demonstrate that the core network acted in the last common ancestor of the mosses and angiosperms that existed sometime before 420 mil- Results lion years ago. Two LRL Genes Are Present in the Genome of the Moss P. patens. The LRL transcription factors belong to group XI of plant bHLH auxin | bHLH | evolution | rhizoids | root hairs transcription factors (18) and share a conserved LRL domain of 36 amino acids between the bHLH domain and the C terminus he evolution of rooting structures was a key morphological (Fig. 1A). To identify LRL homologs in P. patens, we performed Tinnovation that occurred when plants colonized the relatively BLAST searches in selected land plant genomes using the dry continental surfaces of the planet sometime before 470 million AtLRL3 (At5g58010) sequence as a query. Phylogenetic anal- year ago. The rooting structures of the earliest diverging groups of yses identified two LRL genes in P. patens,designatedPpLRL1 land plants comprised systems of rhizoids. Rhizoids are either unicellular filaments (in liverworts and hornworts) or multicel- Significance lular (in mosses) and elongate into the growth substrate or air surrounding the plant. Bryophyte (liverwort, moss, and hornwort) This work describes the discovery of an ancient genetic mech- rhizoids develop on gametophytes, the multicellular haploid stage anism that was used to build rooting systems when plants col- in the life cycle. The evolution of vascular plants was accompanied onized the relatively dry continental surfaces >470 million years by an increase in the morphological diversity of the sporophyte, ago. We demonstrate that a group of basic helix–loop–helix the diploid multicellular stage of the plant life cycle (1). Roots, transcription factors—the LOTUS JAPONICUS ROOTHAIRLESS1- multicellular axes derived from meristems with protective caps, LIKE proteins—is part of a conserved auxin-regulated gene were a key innovation that first appeared in the fossil record network that controls the development of tip-growing cells with ∼ — 380 million years ago and likely evolved at least twice at least rooting functions among extant land plants. This result suggests once among the Lycophytes and at least once in the Euphyllo- that this mechanism was active in the common ancestor of most phyte clade (2). Roots generally grow into the soil and expand land plants and facilitated the development of early land plant – the plant soil interface into different soil horizons. For example, filamentous rooting systems, crucial for the successful coloniza- some root systems extend deep into the soil (taproots), whereas tion of the land by plants. others proliferate in the nutrient-rich horizons near the soil sur- face. However, unicellular, filamentous protuberances called root Author contributions: T.H.Y.T. and L.D. designed research; T.H.Y.T. and B.C. performed hairs, which are morphologically similar to rhizoids, develop on research; T.H.Y.T., B.C., and L.D. analyzed data; and T.H.Y.T., B.C., and L.D. wrote all roots with few exceptions (2–5). Despite the different contexts the paper. in which they develop, root hairs and rhizoids carry out similar The authors declare no conflict of interest. rooting functions, including nutrient uptake and anchorage (6, 7). This article is a PNAS Direct Submission. Group VIII basic helix–loop–helix (bHLH) transcription fac- Freely available online through the PNAS open access option. tors encoded by ROOT HAIR DEFECTIVE SIX-LIKE (RSL) 1To whom correspondence should be addressed. Email: [email protected]. genes positively regulate root-hair development in the angio- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sperm Arabidopsis thaliana and both rhizoid and caulonema in 1073/pnas.1416324112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1416324112 PNAS | Published online July 6, 2015 | E3959–E3968 Downloaded by guest on September 25, 2021 Fig. 1. (A) Schematic representation of gene structure and amino acid alignment of LRL transcription factors. Only the bHLH and LRL domains are shown. (B) Tree of LRL proteins and related groups of bHLH proteins based on ML statistics. This tree resolved PpLRL1, PpLRL2, OsRHL1, AtLRL1, AtLRL2, AtLRL3, AtLRL4, and AtUNE12 within a monophyletic group, which also contains four additional rice and four additional Selaginella moellendorffii genes (Fig. S1). aLRT values are indicated above the nodes. Two other groups of plant bHLHs involved in root-hair and rhizoid development, group VIIIc (1) encoded by the class I RSL genes, and group VIIIc (2) encoded by the class II RSL genes, are labeled in red and green, respectively. The full ML and Bayesian trees are shown in Fig. S1.(C) Unrooted tree showing the relationships of different LRL proteins in land plants using Bayesian statistics. The location of selected LRL proteins from the moss P. patens (Pp), the monocot O. sativa (Os), and the eudicot A. thaliana (At) are indicated in the diagram. The full ML and Bayesian trees are shown in Fig. S2.

(Pp1s173_83V6.1) and PpLRL2 (Pp1s209_22V6.2), respectively of a uniseriate filamentous network (protonema) comprising (Fig. 1B). Maximum- likelihood (ML) analysis resolved PpLRL1, cells with large chloroplasts and transverse cell walls (chlor- PpLRL2, AtLRL1 (At2g24260), AtLRL2 (At4g30980), AtLRL3, onema cells). Subsequently, increasingly longer cells with oblique AtLRL4 (At1g03040), AtUNE12 (At4g02590), five rice proteins cell walls (caulonema cells) develop. Caulonema cells are mor- including OsRHL1 (Os06g08500), and four Selaginella moellen- phologically and genetically similar to rhizoids (19). Both dorffii proteins into a monophyletic group (Fig. 1B, Fig. S1, and PpLRL1 and PpLRL2 were expressed in the central region of the Dataset S1). The LRL domains of PpLRL1 and PpLRL2 are protonema, which is rich in chloronema and from which caulo- 97.2% identical to each other and are 77.8% and 80.6% identical nema develop (Fig. 2 A and B). No expression was detected in to the LRL domain of the AtLRL3 protein, respectively. The two the very edges of the 30-d-old protonema, where caulonema is PpLRL proteins have conserved bHLH domains that are 100% the dominant cell type. These observations suggest that PpLRL1 identical to each other (Fig. 1A). The bHLH domains of the and PpLRL2 are active in cells that develop caulonema. PpLRL proteins are 92.4% identical to the bHLH domain of PpLRL1 promoter was also active in the rhizoid-forming re- the AtLRL3 protein. To infer the phylogenetic relationship of the gion at the base of the gametophore in plants in which the different proteins containing the LRL domain, we constructed PpLRL1 CDS was replaced by the GUS-reporter gene (Fig. 2 C phylogenetic trees using Bayesian and ML methods (Fig. 1C and and D). However, no GUS expression was detected in rhizoids Fig. S2). These trees showed that LRL homologs are present in all themselves. This expression pattern is consistent with a role for 32 land plant species sampled (Fig. S3). The LRL proteins com- PpLRL1 in the regulation of the earliest stages of rhizoid de- prise three major groups, designated XIa, XIb, and XIc (Fig. 1C velopment. PpLRL1 promoter was also active in the gameto- and Fig. S2). Genes that control the development of root hairs are phore apex and leaves, suggesting that this gene likely controls present in group XIa (Fig. 1C, Fig. S2,andDataset S2). PpLRL1 other aspects of gametophore development. PpLRL2 promoter and PpLRL2 are not members of any of these three groups (Fig. activity was detected in the epidermal cells of the gametophore 1C and Figs. S2 and S3). Together, these results indicate that the and leaves (Fig. 2 C and D). Unlike PpLRL1, no GUS activity LRL genes are a conserved group of transcription factors whose was detectable in the buds or at the base of the gametophore. existence predates the divergence of mosses and flowering plants. Like PpLRL1, there was no detectable expression of PpLRL2 promoter in rhizoids. PpLRL Genes Have Different, but Overlapping, Expression Patterns. We hypothesized that LRL genes controlled the development of PpLRL1 and PpLRL2 Are Positive Regulators of Rhizoid Development. rhizoids and caulonemata and that PpLRL1 and PpLRL2 would To determine whether PpLRL genes function in rhizoid de- be expressed in these cell types. To define where LRL genes are velopment, we generated mutants that lack LRL gene function expressed, we replaced the endogenous PpLRL1 and PpLRL2 by homologous recombination (Figs. S5 and S6). To confirm that genes with the coding sequence (CDS) of the UidA glucuroni- homologous recombination had taken place, we characterized dase-encoding gene (GUS) (Fig. S4). Consequently, the GUS the genomic organization of the transformants. PCR analyses expression in these lines identified regions of the protonema in were performed to select transformants in which the endogenous which the PpLRL1 and PpLRL2 promoters are active in re- PpLRL gene was replaced by the gene-targeting construct (Fig. spective loss-of-function Pplrl1 or Pplrl2 mutant background. In S5) and thus represent complete loss-of-function alleles. From P. patens, the germination of spores is followed by the development these, Southern blot analyses were used to further select those

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Fig. 2. GUS staining pattern in plants transformed with PpLRL1pro:GUS and PpLRL2pro:GUS and untransformed controls (WT). (A and B) PpLRL1 and PpLRL2 were expressed in the center of the protonema, composed mainly of chloronema filaments. (C and D) Young buds and gametophores isolated from 4-wk-old protonemata grown on KNOPS-GT medium. PpLRL1, but not PpLRL2, was expressed in the early buds. In the gametophores, PpLRL1 was expressed in the base, where basal rhizoids develop. It was also expressed in the stem and at the apex. PpLRL2, in contrast, was not expressed in the early buds or the stem of the gametophores, but was expressed in the leaves. [Scale bars: 2 mm (A), 1 mm (B and D), and 200 μm(C).]

transformants in which there were no additional transgene insertions mutant plants (Fig. 4 A and B). Caulonemata did not develop, in the genome (Fig. S5). Three independent lines for each gene and, instead, a dense mat of chloronemata constituted the entire were maintained for phenotypic analyses. With the exception of protonema (Fig. 4A). The diameter of the Pplrl1 Pplrl2 double- Pplrl2-1, all single-mutant lines and all three double-mutant lines mutant protonema was approximately half that of the WT, which showed a single band in the Southern blot analysis, indicating can be explained by the absence of caulonemata, because these that there was a single insertion into the genome (Fig. S5). cells have higher growth rates and are longer than chloronemata PLANT BIOLOGY Pplrl1 and Pplrl2 single-mutant plants developed ∼40% fewer (Fig. 4 B and C). To verify that the smaller diameter of Pplrl1 rhizoids than wild-type (WT) (Fig. 3 A and B), and these rhizoids Pplrl2 mutants was due to the loss of caulonemata, we compared were ∼35% and 18% shorter than WT, respectively (Fig. 3C). the growth rate of Pplrl1 Pplrl2 double mutants with WT (Fig. 4C). − Pplrl1 Pplrl2 double mutants were completely rhizoidless, and no In WT plants, chloronema growth rates were 7.94 ± 0.45 μm·h 1, − rhizoids developed on these plants (Fig. 3D). Together, these and caulonemal growth rates were 16.86 ± 0.23 μm·h 1.The data show that the activity of PpLRL1 and PpLRL2 together are growth rates of protonema filaments in Pplrl1 Pplrl2 double − required for the initiation and development of rhizoids from mutants were 8.48 ± 0.81 μm·h 1. That is, the growth rates of buds and gametophores. protonema filaments of the Pplrl1 Pplrl2 double mutant were not To determine whether the bHLH and LRL domains of the significantly different from those of WT chloronema. These data LRL proteins—characteristic of group XI plant bHLHs—are indicate that chloronemata, but not caulonemata, develop in required for the control of rhizoid development in P. patens,we Pplrl1 Pplrl2 double mutants. Pplrl1 and Pplrl2 single mutants generated additional mutants in which the region containing the developed less severe phenotypes than the Pplrl1 Pplrl2 double bHLH–LRL domain of each protein was deleted (Fig. S6). The mutants. Protonema diameters of Pplrl1 and Pplrl2 mutants were organization of the genomic DNA in the mutants confirmed that 83% and 89% that of WT, respectively (Fig. 4 A and B). To- the bHLH–LRL domains were deleted (Fig. S6). These mutants gether, these data demonstrate that PpLRL1 and PpLRL2 pos- were designated Pplrl1ΔbL and Pplrl2ΔbL, respectively. Defects itively regulate the developmental transition from chloronema to in rhizoid and caulonema development in these partial-deletion caulonema during the development of moss protonemata. mutants were similar to the defects in the complete gene- deletion mutants (Fig. 3E). To independently verify that the bHLH Auxin Positively Regulates PpLRL Activity. Auxin positively regu- and LRL domains are positive regulators of rhizoid develop- lates the development of rhizoids and caulonemata in P. patens ment, we overexpressed a fragment of the PpLRL1 and PpLRL2 (8, 9, 20–22). To test whether auxin promotes rhizoid and cau- CDS containing the essential bHLH and LRL domains (Fig. S6). lonema development by positively regulating the expression of Plants were transformed with constructs in which the bHLH– PpLRL genes, we determined the effect of auxin treatment on LRL domain was placed under the control of the CaMV35S the activities of the PpLRL1 and PpLRL2 promoters. P. patens promoter (Fig. S6). Plants transformed with 35S:PpLRL1bL-1, plants in which the endogenous PpLRL CDS were replaced by which overexpressed the bHLH–LRL fragment of PpLRL1, de- the GUS gene (Fig. S4) were grown on Knops-GT medium veloped longer and more intensively pigmented rhizoids than (defined in Plant Material, Growth Conditions, and Phenotypic WT (Fig. 3E). In contrast, rhizoid pigmentation in 35S:PpLRL2bL- Analyses) for 30 d. No GUS expression was detectable in the 1–transformed plants, which overexpressed the bHLH–LRL fragment protonema or buds, even when incubated in glucuronidase re- of PpLRL2, resembled WT (Fig. 3E and Fig. S6). This difference in action buffer for 24 h (Fig. 5A). Addition of 1-naphthaleneacetic rhizoid phenotype in these lines suggests that PpLRL1 plays a acid (NAA) to the medium at a final concentration of 100 nM more important role than PpLRL2 in rhizoid development. resulted in detectable GUS expression in both protonema and Together, these data are consistent with the hypothesis that the buds of PpLRL1pro:GUS and PpLRL1pro:GUS plants (Fig. 5A); bHLH and LRL domains are responsible for PpLRL gene this indicates that exogenous treatment with auxin increases the function in rhizoid development. activity of the PpLRL1 and PpLRL2 promoters. Together, these data suggest that auxin positively regulates PpLRL1 PpLRL Genes Positively Regulate Caulonema Development. The and PpLRL2 expression. transition from chloronema to caulonema, characteristic of WT If auxin positively regulates LRL expression, and LRL genes in protonema development, did not occur in Pplrl1 Pplrl2 double- turn promote caulonema development, we hypothesized that

Tam et al. PNAS | Published online July 6, 2015 | E3961 Downloaded by guest on September 25, 2021 Fig. 3. Rhizoid phenotypes of PpLRL loss- and gain-of-function mutants. (A) Rhizoid phenotypes of complete PpLRL loss-of-function mutants. (Scale bars: 2 mm.) (B) Quantification of rhizoid number. (C) Quantification of rhizoid length. Rhizoid length was determined by measuring the length between the base of the gametophore and the tip of the rhizoid cluster. Both rhizoid number and length were significantly reduced in the Pplrl1 and Pplrl2 single mutants. Rhizoids are absent in the Pplrl1 Pplrl2 double mutant. Asterisks represent P values from two-tailed unequal variance (Welch’s) t tests comparing individual mutant lines with WT. **P < 0.01; ***P < 0.000005. Exact P values are as follows. For rhizoid number, Pplrl1 and WT: P = 2.6 × 10−6; Pplrl2 and WT: P = 3.1 × 10−6;andPplrl1 Pplrl2 and WT: P = 1.5 × 10−8. For rhizoid length, Pplrl1 and WT: P = 1.4 × 10−6;andPplrl2 and WT: P = 1.6 × − 10 3. NA, not applicable (the Pplrl1 Pplrl2 mutant has no measurable rhizoids). Error bars represent SEM (n = 9forWT,n = 15 for Pplrl1, n = 12 for Pplrl2, and n = 11 for Pplrl1 Pplrl2). (D) No rhizoids developed at the base of the Pplrl1 Pplrl2 double-mutant gametophore. (Scale bars: 500 μm.) (E) Phenotypes of PpLRLΔbL partial-deletion mutants and 35S:PpLRLbL mutants. The 35S:PpLRL1bL-1 mutant showed an increased number of rhizoids and increased pigmentation.

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Fig. 4. Protonema phenotypes of PpLRL loss-of-function mutants. (A) Caulonema phenotype of complete PpLRL loss-of-function mutants. Caulonema development was defective in the Pplrl1 and Pplrl2 single mutants, and no caulonemata developed in the Pplrl1 Pplrl2 double mutants. [Scale bars: 2 mm (column 1) and 500 μm (column 2).] (B) Quantification of protonema development in terms of green (chloronema-rich) and nongreen (caulonema-rich) contributions to diameter. Asterisks represent P values from two-tailed unequal variance (Welch’s) t tests comparing individual mutant lines with WT. *P < 0.05; **P < 0.01. The exact P values are as follows. For chloronema, Pplrl1 and WT: P = 0.052; Pplrl2 and WT: P = 0.10; and Pplrl1 Pplrl2 and WT: P = 7.3 × 10−6. For caulonema, Pplrl1 and WT: P = 0.0011; and Pplrl2 and WT: P = 0.074. Error bars represent SEM (n = 8 for WT and Pplrl1; n = 7forPplrl2; and n = 6forPplrl1 Pplrl2). (C) Growth rate of caulonema and chloronema filaments in the loss-of-function mutants. In all of the mutant lines, the growth rate of chloronema filaments was not significantly different from WT, but the growth rate of caulonema was significantly reduced. Asterisks represent the P values from two- tailed unequal variance (Welch’s) t tests comparing individual mutant lines with WT. *P < 0.05; **P < 0.01. The exact P values are as follows: for chloronema: P > 0.25 for all mutant lines compared with WT; for caulonema, Pplrl1 and WT: P = 0.017; and Pplrl2 and WT: P = 0.0024. Error bars represent SEM (n = 3for chloronema; n = 5forPplrl1 and Pplrl2 caulonema; and n = 3 for WT caulonema).

auxin-induced morphological changes in caulonema would re- A–C). This result suggests that auxin-regulated expression of quire PpLRL1 and PpLRL2 activity. To test this hypothesis, we PpLRL1 and PpLRL2 is independent of the STYLISH/SHI compared the morphology of Pplrl1 Pplrl2 double mutants grown transcription factors. in medium containing 100 nM NAA with double mutants grown without added auxin. Pplrl1 Pplrl2 protonema that developed PpLRL1 and PpLRL2 Are Required for Low-Phosphate-Induced in the presence of 100 nM NAA were morphologically identical Development of Caulonema. We hypothesized that PpLRL genes to double mutants grown without auxin (Fig. 5B). That is, Pplrl1 would be required for the adaptive response of protonema to Pplrl2 mutants were resistant to auxin. Furthermore, auxin in- low phosphate; growth of protonema in medium containing duced the development of rhizoids on buds of WT, but rhizoid low phosphate promotes the transition from chloronema to induction did not occur on the buds of Pplrl1 Pplrl2 double caulonema (25). To test this hypothesis, we compared the mutants (Fig. 5C). Together, these data demonstrate that rhizoid phenotypes of Pplrl1 Pplrl2 double mutants with WT in media and caulonema development in plants that lack both PpLRL1 with different phosphate content. Low phosphate promotes the and PpLRL2 function are resistant to auxin. This result is con- chloronema-to-caulonema transition in WT protonema, but the sistent with the model in which auxin-regulated caulonema and protonema morphology of Pplrl1 Pplrl2 double mutants was rhizoid development require PpLRL1 and PpLRL2 activity. identicalinrepleteandnophosphate(Fig.6A and B). That is, The STYLISH1/SHI transcription factors regulate auxin bio- the double mutant protonema was insensitive to changes in synthesis in both A. thaliana and P. patens (23, 24). To test external phosphate concentration, and caulonema did not de- whether auxin promotes the expression of PpLRL1 and PpLRL2 velop. These results suggest that the morphological changes in a PpSHI-dependent fashion in P. patens, we quantified mRNA that take place as part of the adaptive response to low phos- levels in the auxin biosynthesis mutants Ppshi1, PpOXSHI1-5, phate require PpLRL1 and PpLRL2 activity. However, we and Ppshi2. We could not detect a difference in PpLRL1 or could not detect a difference in PpLRL1 and PpLRL2 mRNA PpLRL2 mRNA levels between WT and these mutants (Fig. S7 levels in different phosphate conditions (Fig. S7D), suggesting

Tam et al. PNAS | Published online July 6, 2015 | E3963 Downloaded by guest on September 25, 2021 Fig. 5. Auxin-regulated caulonema and rhizoid development require PpLRL1 and PpLRL2 function. (A) Four-week-old protonema grown with 0 μM(−NAA) or 0.1 μM(+NAA) NAA. Auxin treatment induced the activity of the PpLRL1 and PpLRL2 promoters. [Scale bars: 5 mm (columns 1 and 4) or 500 μm (columns 2, 3, 5, and 6).] (B) The Pplrl1 Pplrl2 mutants are partially insensitive to auxin. (Scale bars: 5 mm.) (C) Close-up of a gametophore from 2-wk-old protonema grown with 0.1 μM NAA. Auxin failed to induce rhizoid development in the Pplrl1 Pplrl2 double mutants. (Scale bars: 500 μm.)

that the induction of caulonemal development in low-phos- Discussion phate media does not increase the expression of LRL genes in The colonization of the land by streptophyte plants sometime P. patens. before 470 million years ago was a pivotal event in the history of the Earth. Structures that anchor plants to their growth substrate PpLRL PpRSL P. patens. and Genes Act Independently in The group and provide access to mineral nutrients, water, and the soil mi- – – VIII basic helix loop helix transcription factors, AtROOT HAIR croflora were key adaptations to life on the relatively dry con- DEFECTIVE6 (AtRHD6) and AtROOT HAIR DEFECTIVE6- tinental surfaces. Recent phylogenetic analyses suggest that the LIKE1 (AtRSL1), promote the development of root hairs rhizoidless algae of the Zygnematales or Colecochaetales (or a by positively regulating AtLRL3 expression (13, 14). To de- clade consisting of Zygnematales and Coleochaetales) are sister termine whether there are regulatory interactions between to the land plants (26–29). If a clade consisting of Zygnematales PpRSL and PpLRL genes, we compared the expression of PpLRL1 and Coleochaetales is sister to land plants, then there are two and PpLRL2 in Pprsl1 Pprsl2 double mutants and WT, and the equally parsimonious models for the evolution of rhizoids, each expression of PpRSL1 and PpRSL2 in Pplrl1 Pplrl2 double mu- involving two changes: (i) rhizoids evolved independently in the tants with WT. We found no evidence of transcriptional regu- common ancestor of land plants and in the more distantly related latory interactions between PpLRL and PpRSL genes in P. patens algal lineages such as the Charales or (ii) rhizoids evolved in the (Fig. S7 E and F). Together, these data suggest that the LRL and common ancestor of Charales, Zygnematales, Coleochaetales, RSL genes are transcriptionally independent of each other in and land plants, but were lost in the common ancestor of the P. patens. Zygnematales and Coleochaetales.

E3964 | www.pnas.org/cgi/doi/10.1073/pnas.1416324112 Tam et al. Downloaded by guest on September 25, 2021 Our results demonstrate that the LRL and RSL genes represent PNAS PLUS core components of a conserved gene-regulatory network that was present in the common ancestor of mosses and angiosperms. However, although we showed that the genetic components of this gene-regulatory network have been conserved, the detailed patterns of transcriptional regulation within this network remain to be resolved. For example, AtRHD6 and AtRSL1 (class I RSL genes) regulate the expression of AtLRL3 in A. thaliana (13, 14), but we could not find evidence that PpRSL genes control PpLRL gene expression in P. patens. If there is a genuine absence of transcriptional regulation between LRL and class I RSL genes in P. patens, then two alternative evolutionary scenarios can be suggested. Either (i) class I RSL genes acquired the ability to regulate the expression of LRL genes in the lineage that gave rise to angiosperm, or (ii) RSL-regulated LRL expression was an ancestral trait that was lost in the moss lineage. The mechanism of auxin-regulated caulonema and rhizoid development in P. patens involves key components of the auxin- signaling pathway that is conserved among land plants (8, 9, 11, 20–23, 31). We showed here that auxin positively regulates the expression of PpLRL1 and PpLRL2 and that the PpLRL genes are necessary for auxin-induced caulonema and rhizoid development. Therefore, the observed GUS activity in the PpLRL1pro:GUS and PpLRL2pro:GUS protonema may be due to higher auxin concentration in the center of the protonema. Previously, it was shown that auxin promotes caulonema and rhizoid development by positively regulating the expression of

the class I PpRSL genes, PpRSL1 and PpRSL2 (8, 9). We showed PLANT BIOLOGY here that auxin positively regulates PpLRL expression. However, because we could not find evidence that class I PpRSL genes Fig. 6. PpLRL1 and PpLRL2 function are required for low phosphate regulate the expression of PpLRL genes, or that PpLRL genes adaptive response. (A) Pplrl1 Pplrl2 double mutants are insensitive to low regulate the expression of PpRSL genes in P. patens, these data phosphate. Protonemata were grown in high (+P) and low (−P) phosphate suggest that auxin positively regulates class I PpRSL and PpLRL medium for 2.5 wk. (Scale bars: 5 mm.) (B) Low phosphate increased cau- genes independently. lonema development significantly in WT protonemata. *P = 0.034 [two- According to the classical P. patens literature (22, 32), game- tailed unequal variance (Welch’s) t tests)] but had no discernable effect on = + tophores form only on caulonema. Our results suggest that bud Pplrl1 Pplrl2 mutants. Error bars represent SEM (n 20 for WT P and Pplrl1 formation can occur in chloronema. The morphology, chloro- Pplrl2 +P; n = 16 for WT-P; and n = 12 for Pplrl1 Pplrl2 −P). plast density, and growth rate of the Pplrl1 Pplrl2 protonema resemble WT chloronema, but buds develop from this mutant. We show here that LRL genes positively regulate the devel- Similarly, the caulonema-less Pprsl1 Pprsl2 loss-of-function mu- opment of the tip-growing rhizoids and caulonema in the moss tants (8, 33) also develop buds from chloronema. Together, these P. patens. LRL genes also positively regulate the development data suggest that the bud-formation process in P. patens is not of root hairs in angiosperms (13–15). Given that similar proteins limited to caulonema, but can develop from chloronema in certain genetic backgrounds. control the development of filamentous rooting cells at the – LRL genes are not general regulators of tip growth. There are plant soil interface in mosses and angiosperms, we conclude that three types of tip-growing cells in P. patens: chloronema, caulo- LRL genes positively regulated the development of tip-growing nema, and rhizoid. The caulonemata are cytologically similar to rooting cells in the common ancestor of mosses and angiosperms rhizoids (33–36). PpLRL genes positively control both rhizoid that existed sometime before 420 y ago (30). and caulonema development, but not chloronema development; Basic helix–loop–helix proteins encoded by RSL genes also chloronema development is indistinguishable from WT in Pplrl1 form a network that controls filamentous rooting cell develop- Pplrl2 double mutants. These results are consistent with the ment. The class I RSL genes, AtRHD6 and AtRSL1, promote observation that caulonemata and rhizoids also have similar re- root-hair development in A. thaliana; root hairs do not develop sponses to auxin and low phosphate, and these responses are on Atrhd6 Atrsl1 double loss-of-function mutants (10). AtRHD6 different from the response of chloronema to auxin (8, 9, 25). and AtRSL1 positively regulate the expression of the class II RSL Together, these data suggest that the PpLRL genes are specific genes AtRSL2, AtRSL3, and AtRSL4 and the LRL gene AtLRL3; regulators of the development of tip-growing cells with a rooting these in turn modulate late stages of root-hair differentiation in function. Similarly, RSL genes are specific regulators of caulonema and rhizoid development and do not regulate chloronema A. thaliana (12). AtRSL4 is a class II RSL gene that is necessary – and sufficient for growth. Modulation of AtRSL4 expression by development (8 10). Therefore, we concluded that LRL and RSL genes specifically regulate rhizoid and caulonema devel- class I RSL genes, environment, and auxin determine root-hair opment and are not simply general regulators of tip growth. This cell size. AtRSL4 controls cell size by controlling the expression conclusion is supported by the finding that the tip-growth de- of genes that encode proteins involved in tip growth (12). Class I fect in Atrsl1 Atrsl2 double mutants is restricted to root hairs in — — RSL genes in P. patens PpRSL1 and PpRSL2 positively reg- A. thaliana; pollen tube development is identical to WT in these ulate rhizoid and caulonema development (8–10). Class II RSL double mutants (10). genes—PpRSL3, PpRSL4, PpRSL5,andPpRSL6—regulate LRL transcription factors are ancient and were present in the caulonema development, but have much more subtle functions last common ancestor of mosses and angiosperms that was extant than class I RSL genes (11). at least 420 million years ago. Bayesian analysis indicates that

Tam et al. PNAS | Published online July 6, 2015 | E3965 Downloaded by guest on September 25, 2021 three monophyletic clades (XIa, XIb, and XIc) constitute the majority of land plant LRL proteins. However, PpLRL proteins are not members of groups XIa, XIb, or XIc. Groups XIb and XIc each contain a single gymnosperm LRL protein that is sister to the other angiosperm LRL proteins in each of these monophyletic groups, suggesting that groups XIb and XIc originated before the divergence of gymnosperms and angiosperms (Figs. S2 and S3). The evolution of group XIa LRL protein is more complex (Figs. S2 and S3), but the presence of a group XIa LRL proteins in Amborella and other angiosperm taxa suggests that this group was present in the common ancestor of extant angiosperms. Only group XIa LRL proteins are known to regulate root-hair development in angiosperms. These results support our conclusion that the role of LRL proteins in regulating filamentous rooting cell development is ancient. Further sampling of LRL genes in well-annotated, high-quality assemblies of mon- ilophyte (ferns and horsetails) and gymnosperm genomes will allow the elucidation of the evolutionary history of LRL genes during land plant evolution. There is evidence that PpLRL1 plays a greater role than PpLRL2 in rhizoid development. First, the PpLRL1 and PpLRL2 expression patterns are different. The strong expression of PpLRL1 in chloronema cells, young buds, and the base of the Fig. 7. Models indicating the transcriptional regulation between auxin, LRL, gametophore is consistent with its role in controlling early stages and class I RSL genes in rhizoid and caulonema development in P. patens (A) of caulonema and rhizoid development. In contrast, PpLRL2 and root hair development in A. thaliana (B). promoter activity was not detected in either young buds or the base of gametophores where rhizoids develop, suggesting that PpLRL2 regulates rhizoid development via a nonautonomous 2.0) with automated parameters (40).The final amino acid alignment, con- mechanism. Second, Pplrl1 mutants have a stronger rhizoid de- taining 164 sequences from 32 species and 121 columns, was used in sub- sequent analyses. This alignment was also used to determine the similarity of fect than Pplrl2 single mutants. Third, overexpression of the the bHLH and LRL domains (as shown in Fig. 1) of selected LRL proteins, by PpLRL1bL partial transcript led to an increased number of using the Ident and Sim program in the Sequence Manipulation Suite (42). rhizoids, whereas overexpression of the PpLRL2bL partial transcript had no discernable effect on rhizoid development. Phylogenetic Analyses. The best-fit model of amino acid evolution for the Nevertheless, the activity of both proteins is required for the trimmed alignments were determined by Prottest (Version 3) (43), and development of rhizoids because only the double mutant does the JTT+Γ (for Dataset S1) or the JTT+Γ+I (for Dataset S2) model was chosen. not form rhizoids. Together these results suggest that, although The ML trees were calculated with PhyML (Version 3.0) (44), with the SH-Like both PpLRL1 and PpLRL2 function in rhizoid development approximate likelihood-ratio test (aLRT) for branch support (45) and the best (because the double mutant does not form rhizoids), PpLRL1 of nearest neighbor interchange and subtree pruning and regrafting for tree improvement. Bayesian analyses were carried out with MrBayes (Ver- plays a greater role than PpLRL2. sion 3.2) (46) for >15 million generations. One tree was saved every 100 generations, and the first 25% of trees were discarded. A 50-majority-rule Conclusion tree was generated from the remaining trees. The default settings were LRL genes are part of an ancient gene-regulatory network that used for gap treatment—i.e., as unknown character in PhyML and as missing controls the development of tip-growing filamentous cells with data in MrBayes. rooting functions—rhizoids, caulonema, and root hairs—at the interface between land plants and the soil. This auxin-regulated Plant Material, Growth Conditions, and Phenotypic Analyses. The Gransden network was active early in land plant evolution and was present WT strain (47) of P. patens (Hedw.) Bruch & Schimp was used in this study. in the last common ancestor of the angiosperms and mosses. This Unless otherwise stated, all plants were grown in a SANYO MLR-351 growth chamber at 25 °C and a 16-h light/8-h dark regime with a pho- − − ancient gene-regulatory network diverged since P. patens and tosynthesis photon flux density of ∼40 μmol m 2·s 1.Plantsweregrown A. thaliana last shared a common ancestor (Fig. 7). on a modified KNOPS medium (referred to as “minimal medium” or “KNOPS-GT”) (47): 0.8 g/L CaN O ·4H O, 0.25 g/L MgSO ·7H O, 0.0125 g/L Materials and Methods 2 6 2 4 2 FeSO4·7H2O, 0.055 mg/L CuSO4·5H2O, 0.055 mg/L ZnSO4·7H2O, 0.614 mg/L Sequence Retrieval and Alignment. To identify potential LRL proteins in H3BO3, 0.389 mg/L MnCl2·4H2O, 0.055 mg/L CoCl2·6H2O, 0.028 mg/L KI, P. patens, we performed BlastP searches using AtLRL3 (At5g58010) amino 0.025 mg/L Na2MoO4·2H2O, 25 mg/L KH2PO4 buffer (i.e., 1.84 mM at acid sequence as a query on Phytozome (Version 9.1) (37) with a E-value pH 6.5 with KOH), and 7 g/L agar (Formedium AGA03). Then, 5 g/L glucose

threshold of 1e-6, and we retrieved all of the resulting sequences. We used and 0.5 g/L ammonium tartrate dibasic (C4H6O6·2H3N) were added as sup- only the primary transcripts where alternative transcripts exist. CDS retrieved plements for routine subculture of macerated protonema tissues (KNOPS were translated to amino acids and then aligned by using Mafft with the medium). Sterile cellophane discs (80-mm 325P Cellulose discs; A.A. Pack- L-INS-I option (38). The alignment was trimmed with TrimAl (Version 1.3) (39) aging) were put on top of the medium to facilitate phenotypic analysis and as implemented in Phylemon (Version 2.0) with automated parameters (40). collection of tissues for subculture. The resulting alignment consisted of 139 sequences and 54 columns from four species. PEG-Mediated Transformation of P. patens. PEG-mediated transformation was To extend our analysis and resolve the relationship among LRL proteins, performed as described (10, 48). Antibiotic selection was performed by sequences were retrieved from BLAST searches on genomes on Phytozome adding 50 μg/mL G418 disulfate or 25 μg/mL hygromycin B to the (Version 9.1) (37), the Pinus taeda v1 transcriptome on the Dendrome project growth medium. (dendrome.ucdavis.edu/resources/blast/), the L. japonicus genome assembly build 2.5 (www.kazusa.or.jp/lotus/index.html), and the Amborella tricho- Southern Blot Analysis. Genomic DNA was extracted from protonema tissues carpa genome (41). Only sequences containing unambiguous LRL domains by using the cetyltrimethylammonium bromide (CTAB) method and then were included. The sequences were aligned with Mafft. The alignment was digested overnight with the appropriate restriction endonuclease. A total of trimmed with TrimAl (Version 1.3) (39) as implemented in Phylemon (Version 1 μg (for Pplrl2, Pplrl1 Pplrl2, and Pplrl2pro:GUS)or2μg (for Pplrl1 and

E3966 | www.pnas.org/cgi/doi/10.1073/pnas.1416324112 Tam et al. Downloaded by guest on September 25, 2021 PpLRL1pro:GUS) of digested genomic DNA, as quantified with a NanoDrop 3-indolyl-β-D-glucuronic acid, and cyclohexyl ammonium salt (X-GlcA; Mel- PNAS PLUS ND1000 spectrometer (Thermo Scientific), was subjected to agarose gel ford) dissolved in N,N-dimethylformamide at 37 °C for 24–48 h. Tissues were electrophoresis before being transferred to a positively charged nylon then destained by incubation in 70–100% (vol/vol) ethanol for at least 24 h. membrane. Southern blots were carried out with the digoxigenin (DIG) No staining was observed in WT plants. system for filter hybridization with DIG-labeled probe generated by PCR according to the DIG Application Manual for Filter Hybridization (Roche). Time-Lapse Microscopy. Filaments at the edge of the protonema growing on DIG-labeled DNA Molecular Weight Marker VII (Roche) was used throughout cellophane disk on minimal medium were photographed every 6 min under a the analysis. Primers for probe synthesis are given in Table S1. Leica DFC310 FX camera mounted on a Leica M165 FC stereomicroscope. Images were analyzed with ImageJ (49). The increase in filament length P. patens Generation of GUS-Reporter Lines. The GUS-reporter lines were every 30 min was used to calculate the growth rate. generated by replacing the endogenous PpLRL1 or PpLRL2 gene with the uidA gene and a hygromycin-resistance cassette from the plasmid pBHrev- qRT-PCR. Ten-day-old macerated protonema growing on minimal medium GUS (pBHSNR-GUS) (9); this strategy for constructing the reporter lines is was used for all qRT-PCRs. Approximately 100 mg of fresh protonema that illustrated in Fig. S4. To generate the GUS reporter lines for PpLRL1, a 871-bp had been gently squeezed to remove excess water was frozen in liquid ni- fragment upstream and a 888-bp fragment downstream of the predicted trogen before RNA extraction with the RNeasy Plant Mini Kit (Qiagen). The PpLRL1 CDS (Pp1s173_83V6) were cloned into the AscI–NcoI and BamHI site, amount of RNA was quantified with a NanoDrop ND1000 spectrometer in the correct orientation, of the pBHrev-GUS plasmid, respectively. Similarly, for the GUS-reporter lines for PpLRL2, a 903-bp fragment upstream and a (Thermo Scientific) or a Qubit 2.0 Fluorometer (Invitrogen) before treatment 880-bp fragment downstream of the predicted CDS (Pp1s209_22V6) were with Turbo DNase (Ambion, Life Technologies). First-strand cDNA synthesis cloned into the AscI–NcoI and BamHI–HindIII site of the pBHrev–GUS plas- was performed with the SuperScript III First-Strand Synthesis System for RT- mid, respectively. These generated the pBHrev-PpLRL1proGUS and pBHrev- PCR (Invitrogen, Life Technologies) by using oligo(dT). The following amount PpLRL2proGUS constructs (Fig. S4). The plasmids were linearized with AscI of DNase-treated RNA was used in the cDNA synthesis: 3.5 μg for detecting (for pBHrev-PpLRL1proGUS) and AscI + HindIII (for pBHrev-PpLRL2proGUS) PpLRL levels in Ppshi1, Ppshi2 and WT; 9.1 μg for detecting PpLRL levels in before PEG-mediated transformation. Stable transformants were selected by PpOXSHI1-5 and WT; 1.3 μg for detecting PpLRL levels in WT treated with PCR analyses and then further selected with Southern blot analysis (Fig. S4). high and low phosphate; 2.7 μg for detecting PpRSL levels in Pplrl1 Pplrl2 Three independent lines, which show the same GUS-staining pattern, were double mutants and WT; and >1 μg for detecting PpLRL levels in Pprsl1 used for each genotype. Pprsl2 double mutants and WT. The resulting cDNA reaction mixture (∼20 μL) was diluted 6.5-fold, and 5 μL of the diluted cDNA mixture was used Generation of P. patens Loss- and Gain-of-Function Mutants. The plasmids directly in a 20-μL qRT-PCR. qRT-PCR was performed with the SYBR Green pBHrev (pBHSNR) and pBNRF (10) were used to generate the loss-of-function PCR Master Mix (Applied Biosystems) and the Applied Biosystems 7300 Real-

constructs in this study. To generate the Pplrl1 complete knockout mutants, Time PCR system. Three biological replicates, each with three technical PLANT BIOLOGY an 812-bp DNA fragment upstream and a 819-bp fragment downstream of replicates, were used for each plant sample. The cycling conditions were as the PpLRL1 were cloned into the SalI–BamHI and MluI–NcoI sites of the follows: 50 °C for 2 min; 95 °C for 10 min; then 40 cycles of 95 °C for 15 s and pBHrev plasmid, respectively (Fig. S5). Similarly, to generate Pplrl2 knockout 60 °C for 1 min. Dissociation curve analyses were performed at the end of mutants, a 697-bp fragment upstream and a 655-bp fragment downstream the cycles to ensure the formation of only a single amplicon for each rep- – – of the PpLRL2 locus were cloned into the SalI BamHI and SpeI AscI site of licate. LinRegPCR (50) was used to estimate the baseline, threshold, and the pBNRF plasmid, respectively (Fig. S5). These generated the pBHrev- primer efficiencies. The starting concentration of the target transcript in a PpLRL1KO and pBNRF-PpLRL2KO constructs. The plasmids were linearized sample, expressed in arbitrary fluorescence units (N ), was computed from with SalI + NcoI (for pBHreb-PpLRL1KO) and SalI + AscI (for pBNRF-PpLRL2KO) 0 the following formula: N = threshold/[(mean efficiency of each primer before PEG-mediated transformation. The Pplrl1 Pplrl2 double mutants were 0 pair)^ Cq] (50). PpGAPDH (11) was used as the internal reference for all ex- generated by retransforming Pplrl1-3 with the pBNRF-PpLRL2KO construct. periments, except for the determination of bHLH–LRL partial transcripts in Stable transformants were selected by PCR analyses and then further selected with Southern blot analysis (Fig. S5). Three independent lines, which showed the 35S:PpLRL1bL and 35S:PpLRL2bL lines, where PpAdePRT was used as the the same phenotype, were used for each genotype. Two independent lines of internal references (51). Primers used are given in Table S1. partial gene-deletion mutants (Pplrl1ΔbL and Pplrl2ΔbL), where only the bHLH and LRL domains are deleted, were generated similarly (Fig. S6) and were Auxin and Low-Phosphate Treatment. For auxin treatments, plants were PCR-verified. grown on KNOPS-GT supplemented with 0, 0.1, or 1 μM NAA dissolved in To generate the 35S:PpLRL1bL and 35S:PpLRL2bL mutants, partial CDS for dimethyl sulfoxide. High-phosphate treatment was carried out by growing the bHLH–LRL region of the PpLRL genes (Fig. S6) were first amplified by RT- plants in KNOPS-GT medium supplemented with 1 mM Mes. The low-phos- PCR and subcloned into the pCR8/GW/TOPO Vector (Invitrogen), then phate medium (−P) was prepared by replacing the KH2PO4 buffer in the

transferred to the p108GW35S vector through the Gateway LR Clonase II KNOPS-GT medium with 0.918 mM K2SO4. system (Invitrogen) for moss overexpression (11). This construct includes the 108 locus to facilitate insertion into the neutral 108 locus in the P. patens ACKNOWLEDGMENTS. We thank Dr. Katarina Lundberg from Prof. Eva genome. The levels of the PpLRL1 and PpLRL2 bHLH–LRL partial transcript Sundberg’s group (Swedish University of Agricultural Sciences) for providing in 35S:PpLRLbL lines were determined by quantitative reverse transcrip- the Ppshi1, Ppshi2, and the PpOXSHI1 lines (23); Dr. Krzysztof Szczyglowski tion-polymerase chain reaction (qRT-PCR) (Fig. S6). (Agriculture and Agri-Food Canada) for providing the Atlrl mutant lines for observation; Nuno Pires for technical assistance and scientific input at the beginning of the project; and Helen Prescott and Lida Chen for laboratory GUS Staining. Unless otherwise stated, protonema inocula were grown on support. T.T.H.Y. was supported by a Clarendon Fund Scholarship and KNOPS medium with cellophane for 2.5 wk and then directly incubated in the Overseas Research Students Awards Scheme; B.C. and L.D. were sup- GUS staining solution: 100 mM NaH2PO4 (pH 7.0 with NaOH), 0.5 mM K3Fe ported by the PLANTORIGINS Marie Curie Network and the EVO500 ERC- (CN)6, 0.5 mM K4Fe(CN)6·3H2O, 0.05% Triton X-100, 1 mM 5-bromo-4-chloro- Advanced Grant.

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