M. polymorpha LATERAL ORGAN SUPPRESSOR 1 specifies lateral organ identity during vegetative growth

We isolated two mutants, vj99 and vj86, that produced abnormal green outgrowths from a population of 105,000 transfer DNA (T-DNA)-transformed M. polymorpha (Fig 1D–1G; S1A and S1B Fig). vj99 and vj86 thalli were hyponastic, bending upward at the thallus margins, unlike wild type (WT) (Fig 1D and 1E; S1C and S1D Fig). A single T-DNA was inserted into the gene Mapoly0028s0118 in vj99 and vj86, suggesting that defective function of Mapoly0028s0118 was responsible for the green outgrowths (S1E Fig). To test this hypothesis, we generated independent mutations in the Mapoly0028s0118 gene by homologous recombination. Mutants of Mapoly0028s0118 generated by targeted deletion developed similar phenotypes to those of the vj99 and vj86 mutants (S1F and S1G Fig). To verify that a defect in Mapoly0028s0118 was responsible for the green outgrowth, we transformed mutant vj99 with a genomic fragment that includes the full-length Mapoly0028s0118 gene. Transformation of the Mapoly0028s0118 genomic fragment into vj99 mutants restored WT development, demonstrating that loss of Mapoly0028s0118 function causes the vj99 phenotype (S1H–S1K Fig). Phylogenetic analysis indicated that Mapoly0028s0118 belongs to the Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1 (ALOG) protein family (S1L Fig). The proteins in this family contain a DNA-binding domain with weak transcriptional activity [27–29]. We named this gene M. polymorpha LATERAL ORGAN SUPPRESSOR 1 (MpLOS1). In addition to the abnormal green outgrowths, gemma cup spacing was abnormal in the Mplos1-1 (vj99) mutant; the distance between neighboring gemma cups was much shorter than in the WT (Fig 1F and 1G).

To more precisely define the nature of the green outgrowths, we performed a phenotypic analysis of the Mplos1-1 mutant. Outgrowths emerged from the ventral side of the thallus near the thallus margins and extended beyond the thallus margin in the Mplos1-1 mutant (Fig 2A–2D). These outgrowths resembled ventral scales in a number of ways. They developed in pairs near the apical notch (Fig 2E and 2F; S2A and S2B Fig). They were in general a single cell layer thick, although the outgrowths located near the apical notch occasionally consist of several cell layers (Fig 2G–2I). Outgrowths located near the apical notch also tended to pile up on one another at the edge of the ventral surface (Fig 2G and 2I). Furthermore, although outgrowths developed, no ventral scales formed on Mplos1-1 mutants (Fig 2C and 2D), suggesting that the outgrowths are modified ventral scales. Taken together, these data suggested that the outgrowths formed from the ventral thallus on Mplos1-1 mutants were related to ventral scales.

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Fig 2. MpLOS1 functions are required for specification of lateral organs.

(A-D) SEM images of WT (A and C) and Mplos1-1 mutant (B and D) thalli. Dorsal (A and B) and ventral (C and D) sides of thalli are shown. Ventral scales in (C) and greenish outgrowth in (D) are highlighted in yellow and red, respectively. (E-I) LSFM images of WT (E and G) and Mplos1-1 mutants (F, H, and I). Observation of apical notch regions from the ventral side (E and F) revealed the role of MpLOS1 in specifying lateral organs as ventral scales (F). Vertical longitudinal optical sections (G-I) around apical notches identified accelerated cell division of lateral organs in Mplos1-1 mutants (H and I). Lateral organs are indicated by arrows in (E and F) and highlighted in red or blue in (G-I). (J and K) Images of ventral scales in WT (J) and the corresponding tissues in Mplos1-1 mutants (K). Note that ventral scale cells are transformed into green tissues that lack mucilage hair cells in Mplos1-1 mutants. Mucilage hair in WT is indicated by an arrow in (J). (L and M) FESEM images of ventral scale cells in WT (L) and the corresponding cells in Mplos1-1 mutants (M). Three independent gemmalings were analyzed in WT and in Mplos1-1 mutants, and in total, 97 and 262 chloroplasts were analyzed in WT and in Mplos1-1 mutants, respectively. Note that ventral scale cells are transformed into photosynthetic cells in Mplos1-1 mutants. Scale bars = 1.5 mm in (A-D), 150 μm in (E and F), 300 μm in (G-I), 200 μm in (J and K), and 2 μm in (L and M). LSFM, light sheet fluorescence microscopy; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; FESEM, field emission scanning electron microscope; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000560.g002

Although similar to ventral scales, these outgrowths differ in a number of characteristics. The abnormal outgrowths formed in Mplos1-1 mutants were greener than typical ventral scales (Fig 2J and 2K). There were more cells in outgrowths than in WT scales (Fig 2G–2I; S2C–S2F Fig). Moreover, the mutant chloroplasts were larger than in WT, and there were more thylakoid membranes in the mutants than in WT (Fig 2L and 2M; S2I and S2J Fig). Rhizoids never differentiated in the green outgrowths of the Mplos1-1 mutants, unlike those in WT ventral scales (S2G and S2H Fig). Taken together, these observations indicated that MpLOS1 plays crucial roles in specifying lateral organs as ventral scales, in which MpLOS1 inhibits cell division and chloroplast differentiation.

MpLOS1 activity is required for the maintenance of meristem activity

The WT thallus comprises an apical–basal axis produced by the activity of apical stem cells. The thallus undergoes periodic bifurcation, and gemma cups develop along the midline of the dorsal surface. When the WT thallus bifurcates, a notch containing an apical stem cell forms on each of the two new apical–basal axes (Fig 3A). This process, the duplication of apical notches and the subsequent growth of thalli, is termed “branching.” Upon branching, adjacent apical notches are initially pushed away by the growth of tongue-like tissues, called central lobes, and subsequently further separated concomitant with the growth of the thallus (Fig 3A) [30]. Gemma cups initiate from dorsal merophytes, clones derived from the cell that are cut off from the dorsal surface of the apical cell [17], and they are regularly spaced along the dorsal midline of each thallus.

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Fig 3. Loss-of-function mutant of MpLOS1 displays defects in meristem maintenance.

(A) Diagrammatic representation of thallus shape transition in M. polymorpha thallus development. Note that distances between duplicated apical notches as well as gemma cups are gradually increased along with the progression of branching. Red squares and black circles indicate apical cells and gemma cups, respectively. Apical–basal axes formed in thalli are indicated by green arrows. (B and C) Apices stained by proMpYUC2:GUS in 10-day-old gemmalings in WT (Tak1) (B) and Mplos1-1 mutants (C). Arrowheads indicate GUS staining at apical notches. (D) The number of apices stained by proMpYUC2:GUS in Mplos1-1 mutants as compared with Tak1 throughout 14 days of gemmaling growth. The structure of a gemma is symmetrical, with a single notch on either side. The number of apices originating from one side of each gemma (hereafter referred to as a “half gemmaling”) was counted. Each time point indicates the mean ± SD. At least 14 gemmalings were analyzed at each time point. p-Values lower than 0.01 were indicated by asterisks (*). (E-G) Cell division activities in Mplos1-1 mutants decreased after 3 days’ incubation. EdU-positive signals of 4-day-old gemmalings in Tak1 (E) and Mplos1-1 mutants (F) are shown in green. (G) EdU uptake activities of Tak1 and Mplos1-1 mutants at the indicated time points. EdU-positive signals detected within half gemmalings were counted. Each time point indicates the mean ± SD. At least six gemmalings were analyzed at each time point. p-Values lower than 0.01 are indicated by asterisks (*). (H-K) Defective apical meristem structures in Mplos1-1 mutants. A single apical cell, as seen in 3-day-old Tak1 gemmalings (H), was not observed in Mplos1-1 mutants (I). GUS staining of proMpYUC2:GUS gemmalings in the apical notch region was broader in Mplos1-1 mutants (K) as compared with Tak1 (J). PMs in (H) and (I) were labeled by proMpEF:LTI6B-GFP constructs. Asterisks indicate wedge-shaped cells that are either apical cells or lateral merophytes. Scale bars = 500 μm in (B and C), 200 μm in (E and F), 20 μm in (H and I), and 100 μm in (J and K). Underlying data for this figure can be found in S2 Data. EdU, 5-ethynyl-2′-deoxyuridine; GFP, green fluorescent protein; GUS, β-glucuronidase; LTI6B, LOW TEMPERATURE INDUCED PROTEIN 6B; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; PM, plasma membrane; proMpEF; promoter M. polymorpha elongation factor-1 alpha; proMpYUC2, promoter M. polymorpha YUCCA2; Tak1, Takaragaike-1; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000560.g003

Gemma cups were more densely arranged along the dorsal surface of Mplos1-1 mutants than in WT (Fig 1G). This suggested that the mutants had defects in gemma cup differentiation or axis development, or both. To address whether MpLOS1 is involved in bifurcation or gemma cup differentiation, we analyzed the number of apices and gemma cups in Mplos1-1 mutants during cultivation. To count meristems, we imaged expression of the promoter M. polymorpha YUCCA2: ß-glucuronidase (proMpYUC2:GUS) construct, which is preferentially expressed in notches [31]. The number of apical notches expressing GUS was not significantly different between WT and Mplos1-1 mutants until day 7 of cultivation, although subsequently, fewer GUS-expressing apical notches were detected in the Mplos1-1 mutants compared with WT (Fig 3B–3D). This indicates that bifurcation occurs normally, at least in the early stages of development. In contrast, the density of apical notches in Mplos1-1 mutants was higher than WT at 3 weeks (S3A and S3B Fig). Importantly, there is no clear difference in the number of gemma cups between WT and Mplos1-1 mutants at day 17 of cultivation, although subsequently, fewer gemma cups with higher density were formed in Mplos1-1 mutants (S3B and S3C Fig). These data suggested that the onset of bifurcation, as well as gemma cup differentiation, is not affected and that the separation process of each apical notch is compromised in Mplos1-1 mutants. The lower number of GUS-positive notches and gemma cups in Mplos1-1 mutants after prolonged cultivation may be due to a secondary effect of slow thallus growth or technical limitations of counting densely clustered apices and small-sized immature gemma cups.

The separation of apical notches is dependent on the division and expansion of cells between notches, where central lobes develop (Fig 3A). We reasoned that defective cell division in apical notches and/or central lobes in Mplos1-1 mutant thalli would lead to defects in apical notch separation. We analyzed the cell division activity of Mplos1-1 mutant gemmalings during 7 days’ cultivation by applying a 3-hour pulse of 5-ethynyl-2′-deoxyuridine (EdU), a thymidine analog that is incorporated into cells during DNA replication (Fig 3E–3G) [32]. In early development (days 1–3), the number of cells labeled by EdU was indistinguishable between the WT and Mplos1-1 mutants (Fig 3G). However, beginning from day 3, the incorporation of EdU was lower in Mplos1-1 mutants than in WT, which suggested that cell division is reduced in Mplos1-1 mutants compared with WT (Fig 3E–3G). To determine whether the cell divisions that led to the separation of the apical notches during branching occur in the apical notches themselves or within the central lobes after they emerge, we measured EdU incorporation in these areas during WT development. We found that many cells incorporated EdU in the incipient central lobe as it forms in the apical notch region (S3D Fig). In contrast, very little cell division is found within the central lobe once it has emerged (S3E Fig). This indicates that cells in central lobes are mainly supplied from the apical notches, whereas the contribution of cell division activity within the central lobe for separating duplicated apical notches is low. Altogether, these data suggest that the rate of cell division in the apical notch is lower in the mutant than in WT, leading to an impaired separation of apical notches during branching.

We also compared the cellular organization of apical meristems of Mplos1-1 mutants and WT. The WT apical meristem comprised a single wedge-shaped apical cell and surrounding merophytes (a group of clonally related cells resulting from sequential cell divisions in a single derivative of the apical cell of a meristem), in which the lateral merophytes and the apical cell display identical shapes (Fig 3H) [17]. In contrast, there were many wedge-shaped cells in Mplos1-1 mutants, in contrast to the single apical cell of WT (Fig 3I; S4A–S4H Fig). These defective cell division patterns in Mplos1-1 mutants led to the formation of less strongly invaginated notch regions (Fig 3I; S4A–S4H Fig). Whereas the expression of proMpYUC2:GUS was restricted to a small area of the WT apical notch, staining was more dispersed in Mplos1-1 mutants (Fig 3J and 3K). Occasionally (3 out of 20 gemmalings at 14 days’ cultivation), apical meristems were aborted in Mplos1-1 mutants (S4I Fig, dotted boxes), a phenomenon not observed in WT in our conditions. These data suggest that MpLOS1 is required for the maintenance of apical meristems. These data also support the hypothesis that Mplos1-1 mutants fail to separate apical notches because of defects in cell proliferation in the apical notches. Gemma cup differentiation as well as bifurcation initiate as in WT, but then subsequent defective meristem activity causes defective axis expansion, resulting in the development of a higher density of gemma cups and apical notches in the Mplos1-1 mutant thallus.

MpLOS1 is expressed in lateral organs but not in apical cells

To define the spatial expression patterns of MpLOS1, we established a line that expressed GUS under the control of 5′ and 3′ regulatory elements that were used in the complementation analysis of Mplos1-1 mutants (S1E Fig). In 4-day-old gemmalings, GUS staining was detected in notches and rhizoids (Fig 4A). Weak signal was observed elsewhere in growing thalli (Fig 4B and 4C). The developing ventral scales in the ventral region of the apical notch stained the strongest (Fig 4B–4F). Staining extended over the entire young ventral scale and the basal region of old ventral scales (Fig 4D and 4E). No signal was detected in the oldest ventral scales (Fig 4D and 4E). The expression of MpLOS1 in ventral scales is consistent with the phenotypic defects seen in these organs in the mutant, further strengthening our hypothesis that MpLOS1 is required for the normal development of ventral scales. We also expressed functional proMpLOS1:enhanced green fluorescent protein (eGFP)-MpLOS1 constructs in Mplos1-1 mutants (S1H, S1I and S1K Fig) to analyze the distribution of MpLOS1 protein on a cellular level. eGFP-MpLOS1 protein was preferentially detected in the ventral parts of the apical notch regions (Fig 4G and 4H; S5 Fig; S1 Movie). In particular, stronger signals were detected all over the young ventral scales as well as at the basal region of old ventral scales (Fig 4G and 4H; S5 Fig; S1 Movie). These results further supported a crucial role for MpLOS1 in the specification of lateral organs as ventral scales. However, eGFP-MpLOS1 proteins were not detected in apical cells or lateral merophytes despite the defect in apical meristem morphology and maintenance in Mplos1-1 mutants (Fig 4G–4I). These findings suggest that MpLOS1 mediates the maintenance of apical meristems non–cell autonomously, although we cannot exclude the possibility that MpLOS1 proteins below the level of detection in the apical meristems maintain meristem activity (Fig 4I).

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Fig 4. MpLOS1 regulates lateral organ development cell autonomously and meristem maintenance non–cell autonomously.

(A-C) GUS activity in ventral thalli of 4-day-old (A) and 10-day-old (B and C) proMpLOS1:GUS-expressing gemmalings. (C) is a close-up image of the dotted square depicted in (B). Images were taken from the ventral side. (D-F) Cross section of GUS-stained 10-day-old proMpLOS1:GUS gemmalings. (D and E) are vertical longitudinal sections, and (F) is a vertical transverse section. (E) is a close-up image of the dotted square depicted in (D). “Young” and “old” in (D) indicates the relative age of ventral scales. (G and H) Functional eGFP-MpLOS1 proteins were not detected in apical meristems but were detected in ventral scales and the cells beneath the basal part of ventral scales. Confocal images of a horizontal optical section (G) and a vertical longitudinal optical section (H) of Mplos1-1 mutant gemmalings that express proMpLOS1:eGFP-MpLOS1 constructs are shown. Cell walls were stained by calcofluor. Apical cells are indicated by asterisks and/or dotted yellow lines. (I) Schematic of MpLOS1 protein localization around apical notches. MpLOS1 protein is detected in ventral scales and the cells located around the basal part of ventral scales. Note that MpLOS1 protein is not present in apical cells. Green indicates cells that contain MpLOS1 protein. The apical cell is in pink. Scale bars = 500 μm in (A, B, and D), 200 μm in (F), and 50 μm in (G and H). GFP, green fluorescent protein; GUS, ß-glucuronidase; MpLOS1, M. polymorpha LOS1; ovs, old ventral scale; proMpLOS1, promoter M. polymorpha LOS1; yvs, young ventral scale.

https://doi.org/10.1371/journal.pbio.3000560.g004

MpLOS1 specifies lateral organ identity during reproductive growth

M. polymorpha produces an umbrellalike gametangiophore (antheridiophore or archegoniophore) that bears antheridia or archegonia during reproductive growth [17]. The gametangiophore is a vertically growing thallus branch [17], and we reasoned that gametangiophore development might be defective in Mplos1-1 mutants. The antheridial receptacles of male Mplos1-1 plants were smaller than WT, and unlike in the WT, antheridia were frequently exposed (S6A–S6C Fig). Moreover, the scales on the antheridial receptacles of Mplos1-1 plants were larger than WT (S6D–S6H Fig). MpLOS1 expression was detected in the ventral scales of the antheridiophore, as well as jacket cells, mucilage cells, and throughout the antheridia in plants harboring the proMpLOS1::GUS transgene (S6I and S6J Fig). These observations demonstrate that MpLOS1 regulates ventral scale development by restricting cell division in both the vegetative and reproductive phases.

The archegonial receptacle of female M. polymorpha is highly lobed, with finger-like structures called digitate rays (Fig 5A). The archegonial receptacle lacks the rows of typical ventral scales that develop in antheridiophores. Instead, a pair of specialized scalelike structures called involucres, which are larger than ventral scales, develop between each digitate ray and enclose the archegonia cluster (Fig 5C and 5D) [17]. In female Mplos1-2 mutants (vj86), large leaf-like structures developed as in antheridiophores (Fig 5E). Moreover, more than two involucre-like structures differentiated between each digitate ray (Fig 5C–5F). Importantly, these involucre-like structures resembled ventral scales in their arrangement in several rows (Figs 1B, 1C, 5G and 5H). This suggests that loss of MpLOS1 function results in the transformation of involucres into more scalelike structures. GUS staining was also detected in immature involucres but not in mature involucres in proMpLOS1::GUS archegoniophores (Fig 5I; S6K Fig), accompanied by staining of all parts of the archegonia including eggs, collars, and venters (Fig 5I; S6K and S6L Fig) [17]. These data suggest that ventral scales are transformed into involucres in a MpLOS1-dependent manner upon the transition from vegetative to reproductive growth, in which MpLOS1 inhibits the growth of two rows of ventral scales, resulting in the formation of a single pair of involucres.

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Fig 5. MpLOS1 is necessary for the specification of lateral organ identity during reproductive growth.

(A and B) SEM images of the dorsal side of archegoniophores in WT Tak2 (A) and in Mplos1-2 mutants (B). Note that archegoniophores in Mplos1-2 mutants display shorter, malformed finger-like structures in place of digitate rays. (C-F) SEM images of the ventral side of archegoniophores in WT (C and D) and in Mplos1-2 mutants (E and F). (D) and (F) are magnified images of (C) and (E), respectively. Instead of a single pair of involucres as developed by the WT (C and D), three pairs of membranous structures developed in Mplos1-2 mutants (E and F). Enlarged leaf-like structures (highlighted in green) also developed in Mplos1-2 mutants (E). Involucres in WT and the three pairs of membranous structures in Mplos1-2 mutants are highlighted in yellow, red, or blue. (G and H) Cross sections of archegonial receptacles at regions between the digitate rays in WT (G) and in Mplos1-2 mutants (H). Note the three pairs of ventral scalelike membranous structures in Mplos1-2 mutants. Arrows indicate involucres or ventral scalelike structures. (I) Cross sections of GUS-stained archegoniophores that express proMpLOS1:GUS. GUS activity was detected in immature involucres. Scale bars = 2 mm in (A, B, C, and E), 400 μm in (D and F), and 200 μm in (G, H, and I). GUS, ß-glucuronidase; MpLOS1, M. polymorpha LOS1; proMpLOS1, promoter M. polymorpha LOS1; SEM, scanning electron microscope; Tak2, Takaragaike-2; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000560.g005

Protein function of ALOG proteins are conserved between Marchantia and rice

Mutation of long sterile lemma (G1), a member of the ALOG gene family in rice, results in the enlargement of sterile lemmas, a small leafy lateral organ in the rice spikelet (flower clusters in grass species, basic unit of inflorescence, consisting of one or more flowers), which is interpreted as a homeotic transformation of a sterile lemma into a lemma [29]. Similarly, Mplos1-2 mutants displayed defects in lateral organ specification that we interpret as the transformation of involucres into ventral scalelike structures. To determine whether the M. polymorpha protein could rescue the homeotic transformation of the rice mutant, we expressed MpLOS1 in rice g1 mutants. Expression of MpLOS1 restored the WT short sterile lemma phenotype (Fig 6A–6C). This suggests that the protein functions of the ALOG proteins have been conserved since the time that M. polymorpha and rice last shared a common ancestor, which likely lacked lateral organs. It further suggests that ALOG family proteins were independently co-opted to specify sporophytic function in the lineage giving rise to rice and gametophytic functions in the lineage giving rise to liverworts when each originated the evolutionary novelty of lateral organs.

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Fig 6. Evolutionarily conserved ALOG family proteins in Marchantia and in rice specify analogous reduced lateral organs.

(A-C) Complementation of rice alog mutants with MpLOS1. Phenotypes of a WT (A), a g1 mutant (B), and a g1 mutant expressing the 35Spro::MpLOS1 construct (C) are shown. Scale bars = 2 mm in (A, B, and C). 35Spro, cauliflower mosaic virus 35S promoter; ALOG, Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1; g1, long sterile lemma; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000560.g006

Land plant ALOG proteins regulate lateral organ development and meristem activity

Here, we report the discovery that MpLOS1, a member of the ALOG protein family, controls both lateral organ development and apical meristem activity in M. polymorpha. MpLOS1 represses the growth of different lateral organs, including ventral scales and involucres, and MpLOS1 expression was detected early in the development of these lateral structures. These data indicate that the gene is required for normal lateral organ development. Furthermore, MpLOS1 activity is required for apical meristem maintenance. However, MpLOS1 is not expressed in the apical meristems or surrounding cells. We propose that MpLOS1 cell autonomously regulates lateral organ development but non–cell autonomously regulates apical meristem maintenance (Fig 4I).

The role of ALOG proteins in meristem maintenance is conserved between monocots and dicots. Oryza sativa TAWAWA1(OsTAWAWA1) and Solanum lycopersicum TERMINATING FLOWER (SlTMF) (the tomato TAW1 homolog) proteins, members of the ALOG gene family in rice and tomato, respectively, repress maturation of meristems during reproductive growth [28,33,34]. Although the angiosperm genes control meristem development, neither SlTMF, Arabidopsis thaliana LSH3 (AtLSH3) (the Arabidopsis TAW1 homolog), nor OsTAW1 proteins are expressed in apical meristems. Instead, they are expressed at lateral organ boundaries [27,33,35]. Taken together, these data from a diversity of land plants suggest that although the ALOG genes act cell autonomously during the development of lateral organs, they act non–cell autonomously to control meristem development. It remains unclear how this might operate, but there is evidence from angiosperms that lateral organ development is required for meristem maintenance [9,36,37].

Taken together with our discovery that MpLOS1 is required non–cell autonomously for meristem maintenance in M. polymorpha, this means that the evolutionarily conserved ALOG family proteins control apical meristems in divergent plant lineages, in which the apical meristems are found in different phases of the life cycle. We propose that this mechanism for controlling shoot meristematic activity was already present in the last common ancestor of Marchantia and rice.

Conserved ALOG proteins negatively regulate lateral organ growth

We discovered that MpLOS1 specifies lateral organ identity by negatively regulating the lateral organ outgrowth; involucres are transformed into ventral scalelike structures during reproductive growth in Mplos1-2 mutants (Fig 5G and 5H). The rice homolog, G1, also represses the development of lateral organs to specify the sterile lemmas. Loss-of-function mutations in OsG1 result in the transformation of small sterile lemmas into large lemmas (Fig 6A and 6B) [29]. Similarly, in tomato, Sltmf mutants display a similar transformation, in which sepals develop leaf characteristics [33]. The conserved function of rice, tomato, and M. polymorpha LOS1 homologs suggests that the role of ALOG proteins in repression of lateral organ development is ancient. This functional conservation among divergent taxa of land plants suggests two alternative hypotheses regarding the evolution of lateral organs.

According to the first hypothesis, the common ancestor of liverworts and the seed plants developed lateral organs whose growth was controlled by one or more ALOG genes. Because lateral organs are present in the gametophyte and the sporophyte phase in liverworts and seed plants, respectively, under this scenario, the common ancestor may have had lateral organs in both the gametophyte and the sporophyte phase. These structures would then have subsequently diverged morphologically during the course of land plant evolution. However, this hypothesis is not well supported by the fossil record. The earliest polysporangiophyte and tracheophyte fossils we know of, such as Aglaophyton and Cooksonia, possessed simple axes devoid of lateral organs [20,21]. This suggests that their last common ancestor with the liverworts likewise lacked lateral organs.

An alternative hypothesis, which is in better accord with the fossil record, is therefore that the common ancestor of liverworts and the seed plants lacked lateral organs in the gametophyte and sporophyte phase but possessed an ALOG-mediated mechanism for controlling some other developmental process. The ALOG-dependent growth-repression mechanism was subsequently recruited independently during the evolution of lateral organs in the separate lineages leading to the liverworts and seed plants. If the last common ancestor did not develop lateral organs, and because ALOG function regulates lateral organ development in both liverworts and seed plants, we suggest that ALOG function was recruited independently during the evolution of lateral organs in different lineages of land plants. The original ALOG function prior to these independent recruitment events may have been to control some aspect of apical meristem activities because growth by apical meristems is a shared characteristic of land plants [38] and this ALOG function is found in both liverworts and seed plants. The recruitment of ALOG function during the independent evolution of lateral organs thus provides a molecular mechanism for the convergent evolution of growth repression in lateral organs.

ALOG proteins may mediate diversification of lateral organs during plant evolution

It has been suggested that the repressive activity of the OsG1 gene on growth led to the evolution of the rice spikelet [29]. Loss-of-function Osg1 mutations revert the sterile lemma into a larger leafy structure, which has been interpreted as similar to a hypothetical ancestral structure (Fig 6A and 6B) [29]. According to this model, the formation of a pair of lower lemmas subtending the floret (rice flower surrounded by two bracts; the external lemma and internal palea) was the ancestral state. Then, during the evolution of rice, OsG1 activity was co-opted to repress the development of the lower lemma, which is now much reduced in size in modern rice compared with the ancestral state, resulting in the formation of the sterile lemma. It is formally possible that MpLOS1 may also have played a similar role in the evolution of lateral organs in liverworts. Several liverwort taxa with thalloid form are suggested to have evolved independently from ancestral leafy liverworts, in which leaves are hypothesized to be transformed into nonphotosynthetic ventral scales with reduced growth during this evolutionary transition [18,19]. We found that MpLOS1 is involved in the specification of lateral organ identities by inhibiting cell division and chloroplast differentiation and that the loss of its function leads to the formation of chlorophyll-containing photosynthetic tissues (Fig 2J and 2K). These green appendages are in fact similar to the green-colored photosynthetic scales formed in the Treubiaceae family of liverworts, whose semithalloid form has been interpreted as an evolutionary transition state between the leafy and thalloid form [39–41]. Therefore, MpLOS1 function may also be associated with the evolution of the thallose body form by repressing leaf growth in ancestral leafy liverworts in the same way that OsG1 suppresses lower lemma development during rice spikelet evolution. It is possible that morphological modification of lateral organs is controlled by the spatial and temporal differences in expression levels of ALOG family genes, and this would provide a mechanism for the establishment of morphological diversification in lateral organs that develop on shoots during land plant evolution.

Conclusion

We demonstrate that MpLOS1, a member of the ALOG gene family, plays a role in integrating meristem activity and lateral organ differentiation in M. polymorpha. MpLOS1 acts by repressing lateral organ growth and is required for meristem maintenance. Because ALOG proteins from angiosperms also repress lateral organ growth and are required for meristem maintenance, and these functions were rescued by MpLOS1, we conclude that protein functions of ALOG family proteins are conserved among these taxa and acted in their last common ancestor. We hypothesize that ALOG genes were co-opted to execute the morphological modification of analogous lateral organs during land plant evolution and contributed to the diversification of lateral organs in shoot systems during the course of land plant evolution.

Plant materials and growth conditions

M. polymorpha Takaragaike-1 (Tak1, male) and Takaragaike-2 (Tak2, female) gemmalings were grown for 3–60 days at 22 °C under continuous light on petri plates containing 1/2 Gamborg’s Basal Salt Mixture (B-5) growth medium (pH 5.5) with 1.2% agar (Nacalai tesque, Kyoto, Japan). Transition to reproductive growth was induced through far-red light supplementation [42].

Generation of mutant plants

Knockout mutants of Mplos1-1 (vj99) and Mplos1-2 (vj86) were isolated by a mutant screen of spores from a cross between Tak1 and Tak2 transformed with the T-DNA vector pCambia1300 as previously described [15,43]. Knockout mutants of Mplos1-3 and Mplos1-4 were generated by gene-targeted homologous recombination [44]. Genetic nomenclature is outlined in [45].

Phylogenetic analysis

Phylogenetic analysis was performed as described by Bowman and colleagues [3]. Protein sequences were collected using the Marchantia genome portal site MarpolBase (http://marchantia.info). Multiple sequence alignments were performed using the MUSCLE program [46] contained in the Geneious software (https://www.geneious.com). Gaps were removed by using Strip Alignment Columns in the Geneious package, and phylogenetic analyses were performed using PhyML (http://www.atgc-montpellier.fr/phyml/).

Plasmid construction

For constructing the proMpLOS1:MpLOS1 plasmid that complements the Mplos1 mutants, a MpLOS1 genomic fragment with a 10-kb upstream region and a 3-kb downstream region was amplified by PCR using Prime STAR GXL polymerase (TaKaRa, Shiga, Japan) and subcloned into pENTR/D-TOPO (Thermo Fisher Scientific, Waltham, Massachusetts, United States), which was subsequently integrated into pMpGWB101 by a Gateway LR reaction [47]. pENTR/D-TOPO that included the proMpLOS1:MpLOS1 complement fragment was modified to establish proMpLOS1:eGFP-MpLOS1 and proMpLOS1:GUS plasmids. A PCR-amplified eGFP coding sequence was inserted in frame with the 5′ end of the MpLOS1 coding sequence by the In-Fusion cloning reaction (TaKaRa, Shiga, Japan) to generate the proMpLOS1:eGFP-MpLOS1 plasmid. The coding sequence of MpLOS1 in the proMpLOS1:MpLOS1 complement fragment was replaced with a PCR-amplified GUS coding sequence by the In-Fusion cloning reaction to generate proMpLOS1:GUS plasmids. pENTR/D-TOPO vectors that included proMpLOS1:eGFP-MpLOS1 or proMpLOS1:GUS fragments were subsequently integrated into pMpGWB101 by the Gateway LR reaction. For overexpression of MpLOS1 in rice g1 mutants, a plasmid carrying pro35S::MpLOS1 was constructed. The MpLOS1 genomic coding sequence was amplified by PCR and subcloned into pENTR/D-TOPO, which was subsequently integrated into pGWB2 by a Gateway LR reaction [48].

Histochemical GUS staining

GUS staining was performed as described by Naramoto and colleagues [49], except that 50 mM sodium phosphate buffer was used. Samples were cleared with 70% ethanol and subsequently mounted using a clearing solution (chloral hydrate:glycerol:water, 8:1:2) for direct microscopic observation or dehydrated through a graded ethanol series and embedded in paraffin or Technovit 7100 resin for microtome sectioning.

Plant embedding and sectioning

Plant material was fixed in FAA (45% ethanol:5% formaldehyde:5% acetic acid in water) for embedding in paraffin and Technovit 7100. For paraffin embedding, fixed plant material was dehydrated in a series of ethanol (25%–50%), t-butyl alcohol (10%–75%), and chloroform (20%) solutions and then embedded in Paraplast (McCormick, Baltimore, Maryland, USA). For Technovit 7100 embedding, fixed samples were dehydrated through a graded ethanol series and embedded in Technovit 7100 resin according to the manufacturer’s instructions (Heraeus Kulzer, Hanau, Germany). Embedded samples were sectioned on a rotary microtome into a series of vertical transverse and longitudinal sections (thickness of 8 μm for paraffin and 4 μm for Technovit sectioning). The obtained sections were further treated with neutral red dyes as a counterstain for GUS-stained samples or toluidine blue for the other samples. Multi-Mount 480 solution (MATSUNAMI, Osaka, Japan) or Entellan new (MERCK, Darmstadt, Germany) were used as mounting agents to preserve the samples on the slides.

ClearSee treatment and staining of cell walls

Plants were fixed with 4% paraformaldehyde (PFA) in 1× PBS for 1 hour at room temperature under vacuum. Samples were subsequently washed twice with PBS and transferred to ClearSee solution (10% xylitol, 15% sodium deoxycholate, and 25% urea in water) [50]. ClearSee treatment was prolonged until samples became transparent. Cell walls were stained for 1 hour with 0.1% (v/v) calcofluor or with 0.1% (w/v) Direct Red 23 dissolved in ClearSee. Stained samples were washed for at least 30 minutes with ClearSee solution before observation.

EdU uptake experiments

Gemmalings were incubated in 1/2 B5 medium containing 10 μM EdU (Click-iT EdU Alexa Fluor 488 imaging kit; Thermo Fisher, Waltham, Massachusetts, USA) for 3 hours. Samples were fixed with 4% PFA in 1× PBS for 1 hour under vacuum and then washed three times in PBS. Coupling of EdU to the Alexa Fluor substrate was performed according to the manufacturer’s instructions. Before observations, samples were cleared with ClearSee solution, and cell walls were subsequently stained by Direct Red 23.

Microscopy

Anatomical features were observed with a light microscope (Olympus BX51) equipped with an Olympus DP71, a light sheet microscope (Zeiss Z.1), or a confocal laser scanning microscope (Olympus FV1000 or Zeiss LSM880). For light microscope observations, a PLAPON 2× objective, a UPlanFl 10× objective, or a UPlanFl 20× objective was used. Light sheet microscope observations were conducted using Lightsheet Z.1 detection optics 5× or Clr Plan-Neofluar 20×. For confocal laser scanning microscopy, cell walls stained by Calcofluor or by Direct Red 23 were excited at 405 nm or 543 nm, respectively, whereas GFP and Alexa 488–labeled EdU were excited at 488 nm. Samples were mounted using ClearSee solution and observed with silicon oil objectives. The 3D reconstruction was done by using Imaris software (BITPLANE, http://www.bitplane.com/). High-resolution images showing ultrastructural details were obtained using an SEM (JEOL JCM-6000Plus NeoScope) and an FESEM (Hitachi SU820).

Data analysis and statistics

Four alleles of the Mplos1 mutants, including Mplos1-1, Mpos1-2, Mplos1-3, and Mplos1-4 displayed identical phenotypes in vegetative growth and, thus, Mplos1-1 mutants were used as a representative allele unless otherwise described. All experiments were repeated at least three times. Representative images were used for the preparation of figures. Statistical analysis was conducted using Excel. For comparing two groups, we used the t test to calculate the p-value. The p-values of the relevant experiments are described in the Supporting information (S2 Data). Materials Design Analysis Reporting checklist is described in the Supporting information (S1 MDAR Checklist).