A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs

Satoshi Naramoto; Victor Arnold Shivas Jones; Nicola Trozzi; Mayuko Sato; Kiminori Toyooka; Masaki Shimamura; Sakiko Ishida; Kazuhiko Nishitani; Kimitsune Ishizaki; Ryuichi Nishihama; Takayuki Kohchi; Liam Dolan; Junko Kyozuka

doi:10.1371/journal.pbio.3000560

. 2019 Dec 9;17(12):e3000560. doi: 10.1371/journal.pbio.3000560

A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs

Satoshi Naramoto ^1,^*, Victor Arnold Shivas Jones ², Nicola Trozzi ^1,³, Mayuko Sato ⁴, Kiminori Toyooka ⁴, Masaki Shimamura ⁵, Sakiko Ishida ⁶, Kazuhiko Nishitani ⁷, Kimitsune Ishizaki ⁸, Ryuichi Nishihama ⁶, Takayuki Kohchi ⁶, Liam Dolan ², Junko Kyozuka ¹

Editor: Mark Estelle⁹

PMCID: PMC6901180 PMID: 31815938

Abstract

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, with lateral organs arising independently in different lineages. Vascular plants and bryophytes (basally diverging land plants) develop lateral organs from meristems of sporophytes and gametophytes, respectively. Understanding the mechanisms of lateral organ development among divergent plant lineages is crucial for understanding the evolutionary process of morphological diversification of land plants. However, our current knowledge of lateral organ differentiation mechanisms comes almost entirely from studies of seed plants, and thus, it remains unclear how these lateral structures evolved and whether common regulatory mechanisms control the development of analogous lateral organs. Here, we performed a mutant screen in the liverwort Marchantia polymorpha, a bryophyte, which produces gametophyte axes with nonphotosynthetic scalelike lateral organs. We found that an Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1 (ALOG) family protein, named M. polymorpha LATERAL ORGAN SUPRESSOR 1 (MpLOS1), regulates meristem maintenance and lateral organ development in Marchantia. A mutation in MpLOS1, preferentially expressed in lateral organs, induces lateral organs with misspecified identity and increased cell number and, furthermore, causes defects in apical meristem maintenance. Remarkably, MpLOS1 expression rescued the elongated spikelet phenotype of a MpLOS1 homolog in rice. This suggests that ALOG genes regulate the development of lateral organs in both gametophyte and sporophyte shoots by repressing cell divisions. We propose that the recruitment of ALOG-mediated growth repression was in part responsible for the convergent evolution of independently evolved lateral organs among highly divergent plant lineages, contributing to the morphological diversification of land plants.

Ancestral land plants lacked leaves; instead, these evolved independently in each lineage and were key innovations that allowed the radiation of plants on land during the lower Palaeozoic. This study of the liverwort Marchantia polymorpha reveals that each time they evolved they used the same molecular mechanism to control leaf development.

Introduction

During 470 million years of evolution, the body plans of land plants diversified independently among the gametophyte and sporophyte life stages of different plant groups. In extant bryophytes, early-diverging land plants, the gametophyte is the dominant phase of the life cycle [1,2]. The gametophyte comprises an apical–basal axis with an apical stem cell and forms structures in which gametes develop (antheridiophores and archegoniophores). In contrast, the sporophyte is dominant in extant vascular plants. The sporophyte comprises an axial system (shoots or stems growing along apical–basal axes) that develops from an apical meristem and forms structures in which haploid spores develop. Therefore, in different plant lineages, gametophytes and sporophytes develop axial systems that are produced by apical meristems [3–5].

Extant bryophytes and vascular plants develop lateral organs on gametophytes and sporophytes, respectively. Apical meristems maintain stem cell activity at their center and iteratively generate lateral organs at the meristem periphery. The spatiotemporal differences in cell division and expansion in lateral organs contribute to the morphological diversity of shoot structures in land plants [6–11].

The liverwort Marchantia polymorpha is a bryophyte that forms a gametophyte axis that undergoes indeterminate planar growth in the form of a flattened mat of tissue, called a thallus. The thallus exhibits strong dorsoventrality; gemma cups, gemmae, and air chambers develop on the dorsal side, whereas rhizoids and ventral scales are formed on the ventral side (Fig 1A) [12–17]. Ventral scales cover bundles of rhizoids that run along the underside of the thallus and facilitate water and nutrient transport over the ventral surface of the thallus (Fig 1B) [17]. In the leafy liverworts, photosynthetic leaves arise next to a tetrahedral single stem cell (apical cell). By contrast, M. polymorpha does not develop photosynthetic leaves. Instead, the ventral scales alternately develop on the left and right sides of the wedge-shaped apical cell on the ventral surface in the apical notch near the growing tip of the thallus. The flattened form, single-cell thickness, and bilateral symmetry of the Marchantia scales resemble the leaves in leafy liverworts (Fig 1C) [16,17]. The ventral scales of M. polymorpha are hypothesized to be homologous to the photosynthetic leaves of the basally diverging leafy liverworts [18,19].

Fig 1 — (A-C) Diagrammatic representation of vegetative M. *polymorpha* thallus. Gross morphology (A), ventral side of thallus with ventral scales arranged in three rows on each side of the thallus (B), and vertical transverse section of a notch region (C). Rhizoids are not shown in (B) to clearly visualize ventral scales. The apical cell is shaded in (C). (D-G) Gross morphology of WT Tak1 (D and F) and Mplos1-1 mutant *vj99* (E and G) gemmalings. The 10-day-old gemmalings (D and E) and 3-week-old (F and G) thalli are shown. The dotted square box in (G) that includes the abnormal green outgrowth is enlarged in the image in the top-right corner. Arrowheads indicate abnormal green outgrowths. Note that, unlike WT, Mplos1-1 mutants displayed upward bending of thalli and formed green outgrowths. Scale bars = 1 mm in (D and E) and 0.5 cm in (F and G). Mplos1, M. *polymorpha LATERAL ORGAN SUPRESSOR 1*; Tak1, Takaragaike-1; WT, wild type.

The fossil record indicates that the shoot of the earliest known land plants comprised branching stems without lateral, determinate organs [20,21]. Subsequently, determinate lateral organs, which develop from the sides of apical meristems, evolved. The earliest example of such a lateral organ is the microphyll that developed on the stems of the sporophyte of Baragwanathia longifolia, a lycophyte, which first appears in the fossil record in the late Silurian [22,23]. No lateral organs are known from the gametophytes of early bryophytes from the Silurian or Devonian; these likely arose subsequently and are found in extant bryophytes. The acquisition and modification of different lateral organ types are likely to have been morphological adaptations to the terrestrial environment that increased photosynthetic efficiency, gas exchange, and water transport [7,24–26].

Mechanisms controlling lateral organ development are well described in angiosperms such as rice and Arabidopsis. However, little is known about the mechanisms that regulate lateral organ development among bryophytes. Therefore, we carried out a forward genetic screen for mutants with defective lateral organ development in the liverwort M. polymorpha to define mechanisms that control lateral organ development in this species. Comparing the roles of the genes that control lateral organ development in liverworts and angiosperms allows the identification of mechanisms that were involved in the independent evolution of analogous lateral organs during land plant evolution [7,24–26].

Results

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.

Fig 2 — (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.

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.

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).

Fig 4 — (A-C) GUS activity in ventral thalli of 4-day-old (A) and 10-day-old (B and C) *pro*MpLOS1:*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 *pro*MpLOS1:*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 *pro*MpLOS1:*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; *pro*MpLOS1, *promoter M*. *polymorpha LOS1*; yvs, young ventral scale.

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.

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.

Fig 6 — (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.

Discussion

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.

Materials and methods

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).

Supporting information

S1 Fig. Responsible gene of vj99 mutant encodes an ALOG family protein (related to Fig 1).

(A and B) Phenotypes of vj99 and vj86 mutants. (C and D) LSFM image of gross morphology of WT (C) and vj99 mutant (D) gemmalings. (E) Overview of the functional MpLOS1 construct and the T-DNA insertion mutants isolated by forward genetic screening. The regions 10,152 bp upstream and 1,689 bp downstream of coding sequences were used to express MpLOS1. (F and G) Phenotypic series of Mplos1 knockout mutants. (H-K) Complementation of Mplos1-1 mutants with a functional MpLOS1 construct. The phenotype of Mplos1-1 (I) is complemented by introducing the genomic MpLOS1 fragment (J) as well as the eGFP-fused MpLOS1 genomic fragment (K). (L) Phylogenetic tree of ALOG family proteins in Arabidopsis, rice, and Marchantia. Green, red, and blue symbols indicate ALOG proteins in Arabidopsis, rice, and Marchantia, respectively. Alignment of ALOG family proteins can be found in S1 Data. Scale bars = 1 cm in (A, B, F, G, H, I, J, and K) and 500 μm in (C and D). ALOG, Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1; eGFP, enhanced green fluorescent protein; LSFM, light sheet fluorescence microscopy; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; T-DNA, transfer DNA; WT, wild type.

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S2 Fig. The mutation in MpLOS1 transforms ventral scales into chlorophyll-containing photosynthetic tissues with an increased number of cells (related to Fig 2).

(A and B) LSFM image of gemmalings in WT (A) and in Mplos1-1 mutants (B) observed from the ventral side. Dotted boxes in (A) and (B) that include apical notch regions are shown as close-up images in Fig 2(E) and 2(F), respectively. Ventral scales in WT or their corresponding tissues in Mplos1-1 mutants are indicated by arrows. (C-F) Vertical transverse optical sections of apical notch regions in 4-day-old gemmalings obtained by LSFM. Optical sections in WT (C and D) and in Mplos1-1 mutants (E and F) are shown. Note that the number of cells that make up mucilage and ventral scales increased, and thus, these tissues became larger in Mplos1-1 mutants. (G and H) Images of ventral scales in WT (G) and the corresponding tissues in Mplos1-1 mutants (H). Note that ventral scale cells are transformed into green tissues that lack rhizoids and mucilage hair cells in Mplos1-1 mutants. Rhizoids and mucilage hair in WT are indicated by an arrowhead and an arrow, respectively. (I and J) FESEM images of ventral scale cells in WT (I) and the corresponding cells in Mplos1-1 mutants (J). Images were composites, made up of 25 and 30 individual images (tiles) in WT and Mplos1-1 mutants, respectively. Scale bars = 150 μm in (A and B), 100 μm in (C and E), 200 μm in (G and H), and 10 μm in (I and G). MpLOS1, Marchania polymorpha LATERAL ORGAN SUPRESSOR 1; LSFM, light sheet fluorescence microscopy; FESEM, field emission scanning electron microscopy; WT, wild type.

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S3 Fig. MpLOS1 plays crucial roles in bifurcation (related to Fig 3).

(A and B) Apices stained by proMpYUC2:GUS in 3-week-old gemmalings in WT (A) and Mplos1-1 mutants (B). Arrowheads indicate GUS staining at apical notches. (C) Number of gemma cups in WT Tak1 and in Mplos1-1 mutants. Each bar indicates the mean ± SD. At least seven gemmalings were analyzed at each time point. (D and E) EdU labeling in central lobes. EdU-positive signals of 5-day-old WT gemmalings (D) and that of 9-day-old WT gemmalings (E) are shown. Note that there is little EdU labeling in the central lobes in 9-day-old gemmalings. Arrowhead indicates central lobes. Scale bars = 1.5 mm in (A and B) and 200 μm in (D and E). p-Values lower than 0.01 are indicated by asterisks (*). Underlying data for this figure can be found in S2 Data. EdU, 5-ethynyl-2′-deoxyuridine; GUS, ß-glucuronidase; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; proMpYUC2, promoter M. polymorpha YUCCA2; Tak1, Takaragaike-1; WT, wild type.

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S4 Fig. MpLOS1 plays crucial roles in maintenance of meristem activities (related to Fig 3).

(A-H) Detailed cellular organization of apical notches in 3-day-old gemmalings in WT (A, C, E, and G) and in Mplos1-1 mutants (B, D, F, and H). Horizontal (A and B), vertical transversal (C and D), and vertical longitudinal optical sections (E-H) obtained after the 3D reconstruction of a series of CLSM images were shown. The cells highlighted by orange or cyan in (A) and (B) were sectioned in vertical longitudinal and vertical transversal directions, respectively. The cells highlighted by orange or cyan in (A-H) are identical cells. Cell walls were stained using Direct Red 23. (I) SEM image of Mplos1-1 mutant gemmalings. Thallus regeneration occurs in Mplos1-1 mutants next to aborted meristems. The aborted meristems are indicated by dotted boxes. Scale bars = 30 μm in (A-H) and 1 mm in (I). CLSM, confocal laser scanning microscopy; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; SEM, scanning electron microscope; WT, wild type.

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S5 Fig. Functional eGFP-MpLOS1 proteins were detected in ventral scales and the cells beneath the basal part of the ventral scales (related to Fig 4).

CLSM image of Mplos1-1 mutant gemmalings that express proMpLOS1:eGFP-MpLOS1 constructs. Vertical transverse sections were obtained after the 3D reconstruction of a series of CLSM images. Cell walls were stained by calcofluor. Scale bar = 50 μm. CLSM, confocal laser scanning microscopy; eGFP, enhanced green fluorescent protein; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1.

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S6 Fig. MpLOS1 plays crucial roles in lateral organ differentiation in reproductive growth (related to Fig 5).

(A-F) SEM image of antheridiophores in WT Tak1 (A and D) and Mplos1-1 mutants (B, C, E, and F). The dorsal side (A-C) and ventral side (D-F) of antheridiophores are shown. (C) and (F) are close-up images of (B) and (E), respectively. Some ventral scales are highlighted by colors. Note the exaggerated growth of ventral scales as compared with the size of thalli (F). (G and H) Vertical sections of antheridia in WT (G) and Mplos1-1 mutants (H). Note the extra cell division of misspecified ventral scales in Mplos1-1 mutants. Ventral scales (G) and misspecified ventral scales (H) are indicated by arrows. (I and J) Cross sections of GUS-stained antheridiophores (I) and antheridia (J) that express proMpLOS1:GUS constructs. Note that GUS activities were detected in the ventral scales in WT (I). (K and L) Cross sections of GUS-stained archegoniophores that express proMpLOS1:GUS. Regions that include mature involucres (K) and the whole image of archegoniophores (L) are shown. Note that in contrast to immature archegoniophores, GUS activity was not detected in mature involucres. Scale bars = 2 mm in (A, B, and E), 1 mm in (F), 400 μm in (C), and 200 μm in (G, H, I, K, and L). GUS, ß-glucuronidase; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1; SEM, scanning electron microscope; Tak1, Takaragaike-1; WT, wild type.

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S1 Movie. The 3D reconstruction of the apical notch region by using CLSM data that display eGFP-MpLOS1 and cell walls.

Cell walls were stained by calcofluor. CLSM, confocal laser scanning microscopy; eGFP, enhanced green fluorescent protein; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1.

(MP4)

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S1 Data. Alignment of ALOG family proteins data under fasta format.

ALOG, Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1.

(FASTA)

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S2 Data. Numerical data related to this manuscript.

(XLSX)

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S1 MDAR Checklist. MDAR checklist file related to this manuscript.

MDAR, Materials Design Analysis Reporting.

(DOCX)

Click here for additional data file.^{(33.3KB, docx)}

Acknowledgments

We thank Kei Saito, Eriko Kida, and Kanane Sato for assistance with transformation and microtome sectioning. We also thank Mayumi Wakazaki for preparing samples for electron microscopy.

Abbreviations

35Spro: cauliflower mosaic virus 35S promoter
ALOG: Arabidopsis LSH1 and Oryza G1
AtLSH3: Arabidopsis thaliana LSH3
EdU: 5-ethynyl-2′-deoxyuridine
eGFP: enhanced GFP
G1: long sterile lemma
FESEM: field emission SEM
GFP: green fluorescent protein
GUS: β-glucuronidase
LOS1: LATERAL ORGAN SUPRESSOR 1
LSFM: light sheet fluorescence microscopy
LSH: LIGHT-DEPENDENT SHORT HYPOCOTYLS
LTI6B: LOW TEMPERATURE INDUCED PROTEIN 6B
MpLOS1: M. polymorpha LOS1
OsTAW1: Oryza sativa TAWAWA1
proMpEF: promoter M. polymorpha elongation factor-1 alpha
proMpYUC2: promoter M. polymorpha YUCCA2
SEM: scanning electron microscope
SlTMF: Solanum lycopersicum TERMINATING FLOWER
T-DNA: transfer DNA
WT: wild type

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The funders of this research are the Grants-in-Aid from the Ministry of Education, Culture, Sports and Technology, Japan (https://www.jsps.go.jp/english/e-grants/) (KAKENHI grant numbers 17K17595 for SN, 18H04836 for RN, 17H06472 for KI, and 17H06465 for JK); Newton Abraham Studentship from the University of Oxford, to VASJ; and ERC advanced Grant EVO-500 (250284) (https://erc.europa.eu/funding/advanced-grants) to LD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Biol. doi: 10.1371/journal.pbio.3000560.r001

Decision Letter 0

Roland G Roberts

12 Jul 2019

Dear Dr Naramoto,

Thank you for submitting your manuscript entitled "A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs" for consideration as a Research Article by PLOS Biology.

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PLoS Biol. doi: 10.1371/journal.pbio.3000560.r002

Decision Letter 1

Roland G Roberts

16 Aug 2019

Dear Dr Naramoto,

Thank you very much for submitting your manuscript "A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

You'll see that both of the reviewers are broadly positive about your study, but each raises a number of concerns that should be addressed before we can consider the manuscript further. IMPORTANT: After discussion with the AE, we will not insist on the experiment requested by reviewer #1 in his point 5 (rescue of the rice mutant by MpTAW1) - although this might further strengthen the paper, we believe that it is beyond the scope of the current study.

In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome resubmission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

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When you are ready to submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Roli Roberts

Roland G Roberts, PhD,

Senior Editor

PLOS Biology

*****************************************************

REVIEWERS' COMMENTS:

Reviewer #1:

[identifies himself as Hiro-Yuki Hirano]

Naramoto et al. revealed that the MpTAWAWA1 encoding an ALOG family protein is required for the development of lateral organs in Marchantia polymorpha, and that the function of ALOG genes to repress lateral organ growth is conserved in bryophytes and angiosperms. The authors identified the gene responsible for the morphological defects such as the formation of abnormal outgrowth and hyponasty of the thallus; the gene encoded a protein belonging to the ALOG family, some members of which are involved in organ and inflorescence development in rice. From the close examination of mutant phenotypes, the authors revealed that MpTAW1 is likely to regulate the development of lateral organs, such as ventral scales in the thallus and involucres in archegoniophore and to maintain the activity of the apical meristem in M. polymorph. Because MpTAW1 was expressed in the lateral organ primordia but not in the meristem, the authors concluded that this gene acts cell autonomously on the lateral organ development and non-cell autonomously on meristem activity. In addition, the authors showed that MpTAW1 rescued a mutant phenotype of the rice g1 mutant, suggesting that protein function of ALOG members is conserved in bryophyte and angiosperms. Thus, ALOG genes regulate the lateral organ development in the gametophyte in the bryophytes and in the sporophyte in the angiosperms.

The results and the interpretation are interesting, and this work is likely to contribute the understanding of not only Marchantia development but also Evo-devo studies in plants. However, several concerns are raised, and I hope they are helpful to improve the work.

Major point:

1. ‘MpTAW1 activity is required for….’, line 142-199

I am confused by the description of the results and interpretation in this section. The authors first described that the expansion of the region between the two apical notches (central lobe) is defective, resulting in the high density of the gemma cups. Then the authors examined the incorporation of EdU in the apical meristem to check the apical notch separation, that is, the expansion of the central lobe.

I am wondering if the lobe expansion is a direct consequence of the activity of the apical meristem. The lobe is outside of the apical meristem consisting of the apical cell and merophytes. Thus, I think that the cells in the central lobe probably proliferate independent of the apical meristem activity. In Arabidopsis root, cells divide and proliferate in the division zone for root growth after differentiation of each cells such as epidermal and cortex cells originated from the root apical meristem. I think that the cell proliferation in the central lobe corresponds to that in the cell division zone of Arabidopsis root.

Therefore, to uncover the cause of a reduction in central lobe growth in the Mptaw1 mutant, the authors should examine whether cell division is impaired in the central lobes during its growth. Conversely, if the cells in the central lobes are supplied only from the apical meristem, such data should be shown.

2. Apart from the above comment, I agree that MpTAW1 is required for the activity of the apical meristem, as the authors showed lower incorporation of EdU. An additional examination such as the observation of division pattern of the apical cell in Mptaw1 would bring information that strengthen the authors’ statement in which MpTAW1 is required for the activity of apical meristem.

3. line 248-250

The authors stated that ‘loss of MpTAW1 function results in the homeotic transformation of involucres into more scale-like structures’, and ‘homeotic transformation’ is repeatedly used in DISCUSSION. However, there is little evidence that the abnormal involucres in the Mptaw1 mutant has ventral scale identity. To assert homeotic transformation, more close morphological examination should be added. Otherwise, I suggest that the authors weaken their claim by removing the word ‘homeotic transformation’ . 　

4. The usage of the word ‘axis’

I am confused by the word ‘axis’. First, what does ‘axial systems’ (line 50, 52) mean?

Second, it is also unclear the following words: ‘a bifurcating axis (line 143)’, ‘axis separation (line 148)’, ‘axis elongation (line 150)’, and ‘axis development (line 155)’. The authors should define these words more clearly. Some of the ‘axes’ should be drawn in the Figure 3A, if possible. But I think that the authors can describe this section more clearly (easily to understand) without the use of ‘axis’.

5. The TAW1, a rice ALOG gene, controls the activity of the inflorescence meristem. The authors claim that MpTAW1 is associated with the apical meristem activity. So, I would like to know whether MpTAW1 rescue the phenotype of loss-of-function mutant of TAW1.

6. The ALOG genes in rice were already named in a preceding paper (Yoshida et al., 2009). But the authors used the different names such as TAW2 and TAW3 in Figure S1. The change of the gene names should be avoided, because it brings confusion. The name of MpTAW1 sound a little bit strange, because the function of MpTAW1 is similar to rice G1 rather than rice TAW1. If the authors want to avoid the use of MpG1, it would be good to create a new name from the mutant phenotype because this study started with the identification of morphological mutant.

7. Transformation of rice g1 mutant.

What promoter did the authors use to express MpTAW1 in rice? There is no description about the experimental procedure concerning rice transformation in MATERIALS AND METHODS.

Are any effects other than the repression of sterile lemma growth observed in the transgenic g1 mutant overexpressing MpTAW1? Why the original lemma was not affected by MpTAW1 overexpression?

Minor points:

1. RESULTS should be written in past tense.

2. line 136: ‘a higher photosynthetic activity in this tissue’

Is it possible to lead this discussion from only starch accumulation?

3. ‘Molecular function of ALOG proteins (line 258, 267, 373)’ should be ‘protein function of ALOGs’, because there is no molecular analysis in the experiment using rice.

4. line 312, ‘The rice homolog…the sterile lemmas’

The reference should be cited.

5. Figure 2E:

The organ with arrows appears to be out of focus a little.

6. Figures 4A-C:

Are these images taken from the ventral side? If so, please indicate this.

Reviewer #2:

[also please see attached word doc for formatted version]

This paper presents interesting and significant results on the regulation of lateral appendage development. These results have important implications for our understanding of the evolution of lateral appendages.

The paper is well written and illustrated. The methods and experimental design are appropriate and solid.

I see a minor issue with the statement on thylakoids (line 134): How many chloroplasts did you assess for that, i.e., what kind of numerical data support this statement about thylakoids?

The only other qualm I have, is with the section of the discussion on lines 320-324, concerning the two alternative hypotheses advanced by the authors regarding the evolution of lateral organs. Your first hypothesis is not specific enough and I don’t see how it would work. Here is why: if the ALOG gene controlled the development of lateral organs in the last common ancestor of the liverworts and the seed plants, where did this common ancestor have lateral organs: in the gametophyte, in the sporophyte, in both phases? The latter should have been the case, given that liverworts have lateral appendages in the gametophyte and seed plants have lateral appendages in the sporophyte. In this case we are, consequently, talking about a plant that had lateral appendages in both the gametophyte and the sporophyte phase and was the last common ancestor of liverworts and seed plants. This ancestor is the same as the last common ancestor of liverworts and tracheophytes (or polysporangiophytes). We don’t know what the earliest liverworts looked like, but it is likely that their gametophytes were thalloid (as suggested by their oldest known fossils) and could have had scales as seen in Marchantia. However, the oldest tracheophytes (and polysporangiophytes) had simple sporophyte axes devoid of lateral appendages. On the other hand, according to at least some phylogenies regarding relationships at the base of embryophytes (if not most of those phylogenies), the two ancestors you are talking about in the two hypotheses could have been the same. So, I think the two hypotheses need to be thoroughly reassessed and this part of the discussion needs to be re-written. Maybe the alternative hypotheses resulting from such a reassessment will have to do with whether the common ancestor had lateral appendages or not and in which phase of the life cycle (gametophyte or sporophyte) it had appendages. Furthermore, if ALOG function in lateral organ development was recruited independently in different embryophyte (land plant) lineages (lines 333-334), you may want to consider and speculate on what would have been their shared function (i.e. in some common ancestor) prior to these independent recruitment events.

Other than that, I only have a few terminology, grammar, and wording suggestions:

- Throughout the manuscript: check the grammar – I found some inadvertent plurals

- Line 31 and throughout – Marchantia is a formal genus name and it should italicized

- Lines 37-38 – the wording is a bit counterintuitive if you say that they specify the identity of lateral organs by repressing their growth. Please reword.

- Lines 59-60 – … forms a gametophyte axis that …

- Lines 69-70 – … and bilateral symmetry of the Marchantia scales resemble …

- Line 74 – is this, rather, Kenrick & Crane 1997? Does it not need to be a numbered reference?

- Line 116 – … much shorter than in the WT

- Lines 145-146 – “distance between each apical notch”??

- Lines 147-148, duplication of apical notches and axis separation – why not refer to this as bifurcation or, even better, just simply branching? After all, it is the same type of process as seen in the apical branching of tracheophyte sporophyte axes.

- Line 186 – the term merophyte has a more specific meaning than the general “cells derived from the apical cell”. The cells produced directly by division of the apical cell are designated as apical derivatives. A merophyte is a group of clonally related cells resulting from sequential cell divisions that originate in a single derivative of the apical cell of a meristem.

- Line 188 and elsewhere – triangular is a misleading descriptor of the shape of these cells, at least as illustrated in the figures referenced. Wedge-shaped may be a more accurate term.

- Line 253 – I have never encountered the term collar in describing archegonium parts. Do you mean archegonial neck?

- Lines 260-261 – your wording implies that a spikelet is a unit of a flower, which is inaccurate. Instead, the spikelet is made of one to several flowers. Please reword.

- Line 302 – your wording implies that rice and Marchantia are the earliest land plants. Please reword.

- Line 314 – Similarly, in tomato, Sltmf mutants …

- Line 374 – check wording

Attachment

Submitted filename: NaramotoEtAl2019_PLOS-Bio_review.docx

Click here for additional data file.^{(14.4KB, docx)}

PLoS Biol. 2019 Dec 9;17(12):e3000560. doi: 10.1371/journal.pbio.3000560.r003

Author response to Decision Letter 1

12 Oct 2019

Attachment

Submitted filename: Response to Reviewers_PLOSBiol20191012.docx

Click here for additional data file.^{(48.4KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3000560.r004

Decision Letter 2

Roland G Roberts

25 Oct 2019

Dear Dr Naramoto,

Thank you for submitting your revised Research Article entitled "A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor.

Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by reviewers #1. IMPORTANT: Please also make sure to address the Data Policy and other policy-related requests noted at the end of this email.

We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by each reviewer. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.

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Roland G Roberts, PhD,

Senior Editor

PLOS Biology

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You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

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BLOT AND GEL REPORTING REQUIREMENTS:

For manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

------------------------------------------------------------------------

REVIEWERS' COMMENTS:

Reviewer #1:

[identifies himself as Hiro-Yuki Hirano]

The authors have addressed most of my concerns and revised the manuscript.

Before publication, however, the authors should check more carefully the manuscript to remove inappropriate expression and grammatical errors. In addition, consistency should be maintained throughout the manuscript; eg., the usage of the mutant name as described below. I suggest the manuscript should be checked by a native speaker of English who preferably understand biology. Probably due to the error when the manuscript was converted to PDF file, figure legends are inserted inappropriate positions in the text.

- There are a number of inconsistent usages of mutant names: los1 mutant, los1-1 mutant, or los1 mutants, including the presence or absence of definite article (the). If the authors used one allele of los1 for phenotypic analysis, the name should be used consistently. Otherwise, respective allele names should be used.

Reviewer #2:

[identifies himself as Alexandru M.F. Tomescu]

I am happy with the revisions you have included.

--------------------

PLoS Biol. 2019 Dec 9;17(12):e3000560. doi: 10.1371/journal.pbio.3000560.r005

Author response to Decision Letter 2

8 Nov 2019

Attachment

Submitted filename: SNVJ_Response to Reviewers_PLOSBiol20191030.docx

Click here for additional data file.^{(24.1KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3000560.r006

Decision Letter 3

Roland G Roberts

11 Nov 2019

Dear Dr Naramoto,

On behalf of my colleagues and the Academic Editor, Mark Estelle, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Responsible gene of vj99 mutant encodes an ALOG family protein (related to Fig 1).

(TIF)

Click here for additional data file.^{(3.7MB, tif)}

S2 Fig. The mutation in MpLOS1 transforms ventral scales into chlorophyll-containing photosynthetic tissues with an increased number of cells (related to Fig 2).

(TIF)

Click here for additional data file.^{(4.3MB, tif)}

S3 Fig. MpLOS1 plays crucial roles in bifurcation (related to Fig 3).

(TIF)

Click here for additional data file.^{(3.6MB, tif)}

S4 Fig. MpLOS1 plays crucial roles in maintenance of meristem activities (related to Fig 3).

(TIF)

Click here for additional data file.^{(3.8MB, tif)}

S5 Fig. Functional eGFP-MpLOS1 proteins were detected in ventral scales and the cells beneath the basal part of the ventral scales (related to Fig 4).

(TIF)

Click here for additional data file.^{(506.6KB, tif)}

S6 Fig. MpLOS1 plays crucial roles in lateral organ differentiation in reproductive growth (related to Fig 5).

(TIF)

Click here for additional data file.^{(7.9MB, tif)}

S1 Movie. The 3D reconstruction of the apical notch region by using CLSM data that display eGFP-MpLOS1 and cell walls.

Cell walls were stained by calcofluor. CLSM, confocal laser scanning microscopy; eGFP, enhanced green fluorescent protein; MpLOS1, M. polymorpha LATERAL ORGAN SUPRESSOR 1.

(MP4)

Click here for additional data file.^{(3.4MB, mp4)}

S1 Data. Alignment of ALOG family proteins data under fasta format.

ALOG, Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1.

(FASTA)

Click here for additional data file.^{(3.1KB, fasta)}

S2 Data. Numerical data related to this manuscript.

(XLSX)

Click here for additional data file.^{(18.6KB, xlsx)}

S1 MDAR Checklist. MDAR checklist file related to this manuscript.

MDAR, Materials Design Analysis Reporting.

(DOCX)

Click here for additional data file.^{(33.3KB, docx)}

Attachment

Submitted filename: NaramotoEtAl2019_PLOS-Bio_review.docx

Click here for additional data file.^{(14.4KB, docx)}

Attachment

Submitted filename: Response to Reviewers_PLOSBiol20191012.docx

Click here for additional data file.^{(48.4KB, docx)}

Attachment

Submitted filename: SNVJ_Response to Reviewers_PLOSBiol20191030.docx

Click here for additional data file.^{(24.1KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs

Satoshi Naramoto

Victor Arnold Shivas Jones

Nicola Trozzi

Mayuko Sato

Kiminori Toyooka

Masaki Shimamura

Sakiko Ishida

Kazuhiko Nishitani

Kimitsune Ishizaki

Ryuichi Nishihama

Takayuki Kohchi

Liam Dolan

Junko Kyozuka

Roles

Abstract

Introduction

Fig 1. Illustration of M. polymorpha thallus and the isolation of Mplos1 mutants.

Results

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

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

MpLOS1 activity is required for the maintenance of meristem activity

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

MpLOS1 is expressed in lateral organs but not in apical cells

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

MpLOS1 specifies lateral organ identity during reproductive growth

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

Protein function of ALOG proteins are conserved between Marchantia and rice

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

Discussion

Land plant ALOG proteins regulate lateral organ development and meristem activity

Conserved ALOG proteins negatively regulate lateral organ growth

ALOG proteins may mediate diversification of lateral organs during plant evolution

Conclusion

Materials and methods

Plant materials and growth conditions

Generation of mutant plants

Phylogenetic analysis

Plasmid construction

Histochemical GUS staining

Plant embedding and sectioning

ClearSee treatment and staining of cell walls

EdU uptake experiments

Microscopy

Data analysis and statistics

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Roland G Roberts

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Decision Letter 1

Roland G Roberts

Roles

Author response to Decision Letter 1

Decision Letter 2

Roland G Roberts

Roles

Author response to Decision Letter 2

Decision Letter 3

Roland G Roberts

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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