The MpANT-Auxin Loop Modulates Marchantia polymorpha Development

Abstract

AINTEGUMENTA-LIKE/PLETHORA/BABYBOOM (APB) genes are considered part of the ancestral developmental toolkit in land plants. In Arabidopsis thaliana, these transcription factors are induced by auxin and are primarily expressed in tissues with actively dividing cells, where they play essential roles in organ development. Marchantia polymorpha, a liverwort that diverged from A. thaliana early in embryophyte evolution, possesses a single APB ortholog, MpAINTEGUMENTA (MpANT), encoded in its genome. In this study, we aimed to characterize the function of MpANT. Analysis of a transcriptional fusion line indicates that MpANT is predominantly expressed in the meristematic region. We report that the MpANT promoter region contains several cis-acting Auxin Responsive Elements (AREs) and demonstrate that its expression, which occurs predominantly in meristematic regions, is significantly altered by the addition of exogenous auxin and inhibition of auxin transport. These findings indicate that MpANT acts downstream of Auxin Response Factors (ARFs) and auxin signaling. Analyses of loss- and gain-of-function MpANT alleles highlight the importance of this transcription factor in meristem maintenance and cell proliferation. Additionally, we found that MpANT acts upstream of the auxin transporter MpPIN1 by influencing auxin distribution. Taken together, our findings reveal a feedforward regulatory loop involving auxin, MpANT, and MpPIN1 that is important for Marchantia development.

Keywords: Marchantia; ABP proteins; EvoDevo; PLETHORA; auxin.

FIGURE 1

MpANT transcription is responsive to fluctuating auxin. (A) Conserved domain organization and euANT‐specific motif of A. thaliana and M. polymorpha euANT clade members. (B) Gene model for MpANT with AREs elements demarcated in its promoter region (pink dots) and the target region for editing with CRISPR‐Cas9 (arrowhead). (C) Expression analysis of MpANT:GFP M. polymorpha line. Confocal images show the expression patterns of 4‐day‐old wild‐type gemmae grown in Gamborg's B5 media (mock), or Gamborg media supplemented with either 2,4‐D (10 μM), NPA (10 μM), and NPA + 2,4‐D. Green fluorescence corresponds to GFP signal, purple fluorescence corresponds to Propidium Iodine signal. Insets in all panels are magnifications of the apical notch. Scale bars = 100 μm.

FIGURE 2

MpANT is important for meristem function in Marchantia thalli. Overall morphology of WT, Mpant, and MpANT ^OE thalli. (A) Gemmae morphology, the black dotted lines show a magnification of the apical notch. Black scale bar = 0.2 mm. (B) Thalli of 4 days‐post‐plated (dpp), the black dotted lines show the apical notches and white dotted lines show a magnification of the transition zones. White scale bar = 2 mm. (C) EdU incorporation assay to visualize the proliferative cells in the apical notch and transition zone of 4 dpp thalli. White dotted lines show Edu‐stained cells. BF, Bright field. Edu scale bar = 200 μm. (D) Gemmae area (mm²) of WT, MpANT ^OE , and Mpant (E) Meristem‐like areas (mm²) of WT, MpANT ^OE , and Mpant young thalli. (F) Area of Edu‐positive cells (μm²) in WT, MpANT ^OE , and Mpant young thalli. One‐way ANOVA followed by Tukey's post hoc test compared to WT (*p < 0.05 and **p < 0.01).

FIGURE 3

Plastochron and branching analyses of WT, Mpant, and MpANT ^OE lines. (A) Thalli of 14 dpp. The black dotted lines show a zoomed‐in meristem zone, and the white arrowheads show differences in the notch divergence. (B) Phenotype of a 1‐month‐old WT, Mpant, and MpANT ^OE plants. (C) Area (mm²) of thallus 14 dpp. (D) Normal plastochron and gemmae cup development in 1‐month‐old plants. (E) Plastochron numbers analysis in WT, Mpant, and MpANT ^OE plants at 30 days. (F) Mean of gemmae cup number at 30 dpp. Scale bars = 1 cm. One‐way ANOVA followed by Tukey's post hoc test compared to Tak‐1 (*p < 0.05 and **p < 0.01), n.s. = non‐significant.

FIGURE 4

MpANT regulates the transcription of genes involved in auxin synthesis and auxin transport, including MpPIN1, which is the sole ortholog of long AtPINs. (A) RT‐qPCR analyses of auxin‐related genes and putative MpANT targets at 7 and 14 dpg in WT and Mpant plants. (B) Graphic representation of the hydrophilic loop in AtPIN1 (against which the polyclonal antibody α‐PIN1, used in our study, was generated), comparison with MpPIN1 and the partial deletion of such region Mppin1‐4 mutant. (C) Colocalization of α‐PIN1 in Wt and MpPIN1:MpPIN1‐Citrine line, the white dotted lines show zoom in the apical notch, rhizoids and attachment cells. Colocalization test shows the Persons correlation coefficient (R) of α‐PIN1 and MpPIN1:MpPIN1‐Citrine channels. White scale bar = 100 μm. (D) Zoom in the apical notch, rhizoids and attachment cells, signal of α‐PIN1 and _pro MpPIN1:MpPIN1‐Citrine and Colocalization test. White scale bar = 100 μm.

FIGURE 5

MpANT acts upstream polar localization of MpPIN1 and auxin distribution in Marchantia gemmae. (A) Immunolocalization assays detecting MpPIN1 and IAA in gemmae of WT, Mpant, and MpANT ^OE , the red signal corresponds to α‐PIN1 (Alexa Fluor 594 conjugated secondary antibody), the green signal to free IAA (Alexa Fluor 488 conjugated secondary antibody). The white color in (A) shows the segmentation process of α‐PIN1 signal localization in the central zone cells. (B) Number of segmentation points located in the membrane and the cytosol in each genetic background. One‐way ANOVA followed by Tukey's post hoc test compared to Tak‐1 (**p < 0.01). White scale bar = 100 μm. (C) PIN1 and IAA distributions in apical notch and rhizoid cells. In the notch zone, white arrows show IAA accumulation in the border cell, transitions zone, apical, and sub‐apical cell, respectively. In rhizoid cells, white arrows show the IAA accumulation in the endomembrane and inside the rhizoid cells. White scale bars = 50 μm.

FIGURE 6

The roles of MpANT roles in regulatory networks in the meristem (purple box) and in epidermal cells (pink box) of Marchantia. Diverse studies and ours show the expression of MpANT in the apical notch, coincident with an auxin maximum. Here, we show that increasing auxin concentrations induces MpANT transcription, most probably via ARFs action. MpANT acts upstream of auxin synthesis genes MpTAA and MpYUC2 as well as on MpPIN1 involved in auxin transport. This loop is essential for the proper development of the Marchantia meristem. Additionally, in the epidermal cells, the localization of MpPIN1 to the plasma membrane depends on MpANT, probably by regulating the expression of putative AGC Kinases encoded in the Marchantia genome. The described functions for MpANT in the meristem and epidermal cells are not mutually exclusive.