(A) 10-day-old wild-type gemmaling with four apical notches and well developed air pores. (B) 10-day-old wild-type gemmae grown on 10 μM NAA. (C) Dorsal epidermis of 15-day-old wild-type thallus. (D) Dorsal epidermis of 15-day-old wild-type thallus, transferred on day 6 to 10 μM 2,4-D with abnormal air pores. (E) 23-day-old wild-type thallus with three gemmae cups. (F) 23-day-old wild-type thallus transferred at day 7 to 10 μM NAA with elongated gemmae cup. (G) Wild-type mature gemmae cup. (H) Wild-type mature gemmae cup from 17-day-old plant, transferred at day 12 to 10 μM 2,4-D. (I) Wild-type mature gemmae cup with gemmae inside. (J) Wild-type mature gemmae cup from a plant grown on 100 μM L-Kyn. (K) Ventral side of wild-type thallus grown on mock media. (L) Ventral side of wild-type thallus grown on 100 μM L-Kyn; a decrease in rhizoid number exposes ventral scales. (M) Mature thallus showing a thallus lobe separating two recently bifurcated apical notches. (N) Mature thallus of plants transferred on day 7 to 100 μM L-Kyn, showing a smaller lobe spacing apical notches and fused gemmae cup primordia (arrowhead). (O) Strong _pro EF1:iaaL lines are composed of an undifferentiated mass of cells that fail to establish a clear dorsiventrality. (P) Close up view of _pro EF1:iaaL line shows a mass of undifferentiated cells although rhizoids are able to differentiate. (Q) Expression pattern of _pro MpSHI in mature thallus with staining in the vicinity of the apical cell. (R) Different degrees of gemmae cup fusion observed in 100 μM L-Kyn treated plants. (S) _pro MpSHI:iaaL plants show an irregular zig-zag arrangement of gemmae cups that are in close proximity to each other. Scale bars in A, B, E, F, G, H, J, K, L, O, R and S = 1 mm; C, D and P = 0.6 mm; I = 0.5 mm; M = 1.2 mm; N = 0.75 mm; Q = 0.2 mm. Asterisk, apical notch; a, air pores; gc, gemma cups; g, gemmae.

(A) RLM RACE of amiRMpIAA expressing lines. Left panel shows gel electrophoresis of RLM-RACE products. Right panel shows direct sequencing of the 633bp RLM-RACE product from amiRMpIAA ₉. Sequencing detected the expected cleavage of MpIAA between bases 10/11 of both miR sequences. (B) Auxin hypersensitivity in knock-down MpIAA lines at both the sporeling and adult stages. At the sporeling stage, _pro EF1:amiRMpIAA ₉ sporelings grown on 5μM 2,4-D from germination have severely affected area compared to controls. In the adult stage plants were grown for 16 days on B5 media prior to treatment with 2,4-D. _pro EF1:amiRMpIAA ₉ treated with 7.5μM 2,4-D for ten days form ectopic rhizoids compared to controls. In all panels, each of the plants is an independent transformant. Scale Bars = 1 cm.

(A) Each panel show six independent lines, transformed with constructs as indicated above the panels. Plants are treated as indicated to the left. Arrows indicate apical notches. (B) Left panel: Rhizoids from control Hyg^R lines. Right panel: _pro EF1:MpARF1 ^ΔD34 lines develop elongated and profuse rhizoids. (C) Apical notch and gemmae cup measurements for control, _pro EF1:MpARF1 and _pro EF1:MpARF1 ^ΔD34 primary transformants after 50 days of growth (30 independent lines were scored per genotype). MpARF1 overexpression results in increased branching rates. Deletion of D34^MpARF1 recovers the gemmae cup production lost by over-expressing full length MpARF1 transcripts. (D) Semi-quantitative RT-PCR showing six independent _pro EF1:MpARF1 lines exhibiting an inverse correlation between MpARF1 and MpIAA transcript levels. Three of the six lines are shown in the panel, with line L6 having more extensive branching and a smaller size compared to line L2. The constitutive translation ELONGATION FACTOR 1-alpha (EF1) was used as a control. All scale bars = 1 cm, except D = 1 mm.

(A) Mature dormant gemma of _pro MpTPL:3xVENUSN7 with expression in two apical notches (asterisks). (B) 5-day-old gemmae of _pro MpTPL:3xVENUSN7. Expression has expanded from apical notches (asterisk) to zones of newly formed air chambers (arrowheads). Inset illustrates background fluorescence in the wild type with identical settings. (C) Mature 25-day-old _pro MpTPL:3xVENUSN7 line showing expression in meristematic regions (asterisks). (D) Young gemmae cup (gc) primordia in mature _pro MpTPL:3xVENUSN7 line. (E) Expression pattern of mature _pro MpTPL:3xVENUSN7 thallus on mock and 10μM 2,4-D media. Plants were transferred to exogenous auxin after 16 days on B5 media. (F) _pro EF1:VENUS indicating expression in apical notches (asterisk) and gemmae cups (gc). (G) Dorsal side of wild-type thallus covered with photosynthetic air chambers and two apical notches from which gemmae cups (gc) develop. (H) _pro EF1:MpTPL plants show dorsiventrality but are affected in cup development (arrowhead). (I) Representative _pro EF1:MpTPL ^N176H line with a compact and multi-bifurcated thalli, with at least 7 apical notches (asterisks) produced in a similar area compared to (G). (J) _pro EF1:MpTPL plants have diminished area in low auxin concentrations compared to controls. Each plant is an independent primary transformant grown for a month on 2.5μM 2,4-D. (K) Wild-type mature gemmae cup harboring dormant gemmae. (L) _pro EF1:MpTPL plants have no gemmae cups, instead naked gemmae and gemmae primordia with filaments growing by transversal cell divisions can be seen. (M) _pro EF1:MpTPL ^N176H gemmae cups are elongated and narrow, similar to developmental effects produced by exogenous auxin application in the wild type. Scale bars in A and B = 0.5mm; C, D, E, F, H, M = 1mm; G and I = 1.5mm; J = 1 cm; K = 300μm; L = 75μm; M = 0.6mm.

(A) _pro EF1:MpTPL-D34 ^IAA lines suffer from severe dwarfism compared to _pro EF1:MpTPL plants. (B) Strong _pro EF1:MpTPL-D34 ^IAA line showing a lack of organized patterning, dorsiventral polarity and a limited degree of differentiation. Scales (arrowhead), air pores (asterisk) and rhizoids (r) are visible in random places and growth is severely affected. (C) _pro EF1:MpTPL-D34 ^MpARF1 representative line showing a degree of differentiation similar to the prothallus stage of wild-type development. (D) _pro EF1:MpTPL-D34 ^MpARF1 showing single cell layer prothalli-like tisssue. (E) _pro EF1:MpTPL-D34 ^MpARF2 representative line. (F) Wild-type sporeling (10 days) transitioning into a single cell layered prothallus (14 days) and fully established multiple prothalli 18 days after plating. (G) Strong _pro EF1:MpTPL-D34 ^BDL line lacking growth, differentiation and dorsiventrality, but still capable of producing rhizoids. (H) _pro EF1:MpTPL-D34 ^MpARF3 lines differentiate gemmae cups and a dorsiventral polarity with dorsal air chambers and ventral rhizoids. The thallus has an apparent hyponastic curvature. (I, J) Multiple independent Hyg^Res sporelings (transformed with an empty binary vector) grown on media containing 10μg/ml Hyg and mock (I) or 10μM 2,4-D (J) for 14 days. (K, L) Multiple independent _pro EF1:MpTPL-D34 ^MpIAA (K) or _pro MpTPL:MpTPL-D34 ^MpIAA (L) lines grown on 10μM 2,4-D for 14 days. Scale bars in C, E, I, J, K and L = 1mm; A = 1cm; B = 430μm; D = 130μm; F = 30μm; G = 600μm; H = 860μm.