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