A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha

Abstract

In land plants comparative genomics has revealed that members of basal lineages share a common set of transcription factors with the derived flowering plants, despite sharing few homologous structures. The plant hormone auxin has been implicated in many facets of development in both basal and derived lineages of land plants. We functionally characterized the auxin transcriptional response machinery in the liverwort Marchantia polymorpha, a member of the basal lineage of extant land plants. All components known from flowering plant systems are present in M. polymorpha, but they exist as single orthologs: a single MpTOPLESS (TPL) corepressor, a single MpTRANSPORT inhibitor response 1 auxin receptor, single orthologs of each class of auxin response factor (ARF; MpARF1, MpARF2, MpARF3), and a single negative regulator auxin/indole-3-acetic acid (MpIAA). Phylogenetic analyses suggest this simple system is the ancestral condition for land plants. We experimentally demonstrate that these genes act in an auxin response pathway--chimeric fusions of the MpTPL corepressor with heterodimerization domains of MpARF1, MpARF2, or their negative regulator, MpIAA, generate auxin insensitive plants that lack the capacity to pattern and transition into mature stages of development. Our results indicate auxin mediated transcriptional regulation acts as a facilitator of branching, differentiation and growth, rather than acting to determine or specify tissues during the haploid stage of the M. polymorpha life cycle. We hypothesize that the ancestral role of auxin is to modulate a balance of differentiated and pluri- or totipotent cell states, whose fates are determined by interactions with combinations of unrelated transcription factors.

Fig 1. Developmental effects of auxin in the M. polymorpha thallus.

(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.

Fig 2. MpIAA represses auxin signaling in M. polymorpha.

(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.

Fig 3. Gain-of-function MpARF1 lines are auxin hypersensitive.

(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.

Fig 4. Developmental role of MpTPL in M. polymorpha.

(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.

Fig 5. Developmental effects of auxin insensitivity.

(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.