KANADI promotes thallus differentiation and FR-induced gametangiophore formation in the liverwort Marchantia

Abstract

In angiosperms, KANADI transcription factors have roles in the sporophyte generation regulating tissue polarity, organogenesis and shade avoidance responses, but are not required during the gametophyte generation. Whether these roles are conserved in the gametophyte-dominant bryophyte lineages is unknown, which we examined by characterising the sole KANADI ortholog, MpKAN, in the liverwort Marchantia polymorpha. In contrast to angiosperm orthologs, MpKAN functions in the gametophyte generation in Marchantia, where it regulates apical branching and tissue differentiation, but does not influence tissue polarity in either generation. MpKAN can partially rescue the sporophyte polarity defects of kanadi mutants in Arabidopsis, indicating that MpKAN has conserved biochemical activity to its angiosperm counterparts. Mpkan loss-of-function plants display defects in far-red (FR) light responses. Mpkan plants have reduced FR-induced growth tropisms, have a delayed transition to sexual reproduction and fail to correctly form gametangiophores. Our results indicate that MpKAN is a modulator of FR responses, which may reflect a conserved role for KANADI across land plants. Under FR, MpKAN negatively regulates MpDELLA expression, suggesting that MpKAN and MpDELLA act in a pathway regulating FR responses, placing MpKAN in a gene regulatory network exhibiting similarities with those of angiosperms.

Keywords: Marchantia; KANADI; far-red (FR) responses; gametangiophores; land plant evolution; transcription factor evolution.

Fig. 1

Expression analysis of MpKAN and generation of Mpkan loss‐of‐function plants in Marchantia. (a) MpKAN transcripts were detected via in situ hybridisation in the immediate dorsal and ventral derivatives of apical cell divisions in longitudinal sections of thalli apical notches. Asterisk indicates putative site of apical cell. d., dorsal; v, ventral; ap, air pore. (b–d) Expression patterns of MpKAN as assessed by _pro MpKAN:GUS reporter gene expression in (b) 4‐d‐old gemmalings, (c) archegoniophores and (d) antheridiophores. (b) In 4‐d‐old gemmalings, when apical notches first bifurcated during plastochron 1, expression was detected in the apical notches (asterisks) and the filaments (arrow) of air chambers closest to the apical notch. (c, d) Reporter gene expression was observed in archegoniophores (c) and antheridiophores (d), predominantly in receptacle tissues. (e) Schematic diagram depicting Mpkan gene structure, domain‐encoding sites and gRNA sites for mRNA sequence of wild‐type and loss‐of‐function alleles. Mpkan‐5^ge and Mpkan‐7^ge harbour deletions at gRNA1 and gRNA2 targeting sites. (f) Raw RNA‐seq density reads (Sashimi plots) of wild‐type and Mpkan lines at the MpKAN locus. Wild‐type MpKAN mRNA consists of six exons, whereas Mpkan alleles resulted in loss of expression of exons 2–5. WT (g–h), Mpkan (i–j) and Mpkan‐5^ge × _pro MpKAN:MpKAN‐CDS (complemented) (k, l) plants grown for 7 d from gemmae. Asterisks indicate apical notches. (m) Wild‐type, Mpkan and complemented lines had largely similar thallus areas. Complemented lines showed restored branching and air pore numbers (n, o), but not gemmae cup numbers (p). (m–p) Data are mean ± SD. Statistical differences among genotypes were determined using one‐way ANOVA and Tukey’s multiple comparisons tests, with letters indicating statistically significant groups (P < 0.01). Where present, numbers after genotype indicate independent lines. Bars: (a) 125 μm; (b–d) 100 μm; (e) 500 bp; (g–l) 2 mm.

Fig. 2

Loss of MpKAN activity affects growth and differentiation in the vegetative gametophyte in Marchantia. (a, b) Scanning electron micrographs of dorsal tissues in wild‐type (a) and Mpkan‐5^ge (b) gemmalings. (c–f) Light microscopy images of wild‐type (c, d) and Mpkan (e, f) gemmalings. (a–f) Asterisks indicate apical notches, and arrows, outgrowths from central zone. (g) Differentiation area index values of wild‐type and Mpkan lines. Statistical differences among genotypes were determined using one‐way ANOVA and Tukey’s multiple comparisons tests, with letters indicating statistically significant groups (P < 0.001). (h) Timing of completion of the first three branching plastochrons in wild‐type and Mpkan plants. Statistical differences were calculated using one‐way ANOVA and Tukey’s multiple comparisons tests. P‐values: **, <0.01; *, <0.05; ns, nonsignificant. (g, h) Data are mean ± SD. (i–l) Morphology of 24‐d‐old wild‐type and Mpkan thalli. (i, j) Wild‐type plants have entered plastochron 4 and have produced two series of gemmae cups originating during plastochrons 2 and 3. (k, l) Mpkan plants are further progressed into plastochron 4; however, gemmae cups have only emerged during plastochron 3. Bars: (a, b) 500 μm; (c–f) 1 mm; (i–l) 5 mm.

Fig. 3

Mpkan mutants fail to complete far red (FR)‐induced developmental transitions in Marchantia. (a) θ _TS values of 5‐d‐old gemmalings grown under constant WL (solid bars) and FR (striped bars). Data are mean θ _TS (columns) ± SD. Statistical differences obtained using two‐way ANOVA and Tukey’s multiple comparisons tests. Letters denote statistically significant different groups (P < 0.05). (b) Ratio of receptacles : apical notches in wild‐type and Mpkan FR‐induced plants. Data are mean ± SD. (c–h) Comparison between the sexual tissues of wild‐type, Mpkan and complemented lines induced under FR‐enriched light conditions. Plants were grown under FR from gemmae for 35 d (c–e, g, h) and 45 d (f). (i–q) SEM imaging of dorsal (i, j, m–o) and ventral (k–l, p–q) reproductive tissues (*, other positions where archegonia are evident). Wild‐type male (i, k) and female (m, p) and Mpkan male (j, l) and female (n, o, q) thalli were grown for 55 d under FR. Magnified insets depict (o) archegonia and (p) involucres. Panels (i–n) and (p) are composites of SEM images. Bars: (c–h, i–n, p) 1 mm; (o, q) 500 μm.

Fig. 4

DEX‐inducible overexpression of MpKAN and analysis of transcriptional targets of MpKAN in Marchantia. (a) Phenotypes of wild type (left) and _pro MpEF1:MpKAN‐GR (right) transformants treated with mock (top) and 10 μM DEX (bottom). Plants were grown for 9 d. (b) Comparison of angle between thallus and substrate (θ _TS) of mock and DEX‐treated plants. Data are mean (columns) ± SD. Statistical differences were obtained using two‐way ANOVA and Tukey’s multiple comparisons tests. Letters denote statistical significant differences (P < 0.05). (c, d) TPM plots of (c) MpTAA and (d) MpDELLA loci from transcriptome profiles of white light (WL)‐ and far red (FR)‐treated wild‐type and Mpkan plants. Values associated with brackets are logFC values (adj. P < 0.001). (e‐h) Volcano plots showing FR‐upregulated and FR‐downregulated genes in wild‐type (e, f) and Mpkan (g, h) transcriptomes. LogFC values obtained using the edgeR software comparing wild‐type FR with wild‐type WL (e, males; f, females) and Mpkan FR with Mpkan WL (g, males; h, females). Yellow loci, nonsignificant (adj. P < 0.001); grey loci, −1 < logFC < 1; blue loci, FR‐upregulated (logFC > 1) or FR‐downregulated (logFC < −1) genes. Transcriptional regulators, peptides/receptors, and phytohormone‐ and light‐related genes (Bowman et al., 2017) were annotated on volcano plots. Genes of interest with logFC > 1 or logFC < −1 are labelled in black. Genes of interest in teal showed same FR response in all sexes and genotypes. Genes of interest in pink showed same FR response in both sexes for one genotype, but not in the other genotype (and are shown in red text in volcano plots of other genotype). Bars, (a) 1 mm.

Fig. 5

MpKAN can rescue the polarity defects of kanadi mutants in Arabidopsis. Comparison between leaves (a) and bolting plants (b) of representative wild‐type, kan1‐2 kan2‐1/+, kan1‐2 kan2‐1 and pAtKAN1:MpKAN kan1‐2 kan2‐1 long‐day grown plants. (a) Detached 6^th leaf of each of the four genotypes with the adaxial surface facing upwards, with the exception of the kan1‐2 kan2‐1 leaf, which is positioned on its side with abaxial (ab), and adaxial (ad) surfaces indicted. kan1‐2 kan2‐1/+ and pAtKAN1:MpKAN kan1‐2 kan2‐1 leaves can be seen to curl inwards relative to wild type but lack the pronounced abaxial outgrowths and reduced expansion of kan1‐2 kan2‐1 leaves. (b) 7‐week‐old bolting plants with the exception of kan1‐2 kan2‐1, which bolt later because of growth disruptions to the inflorescence stem. Some perturbance in kan1‐2 kan2‐1/+ and pKAN1:MpKAN kan1‐2 kan2‐1 inflorescence growth is apparent as twisted stem in axillary shoots (arrowheads). Bars: (a) 5 mm; (b) 1 cm.

Fig. 6

Theoretical pathways for the regulation of far‐red (FR) responses by KAN transcription factors (TFs) in land plants. Components with yellow background are proposed to be conserved in land plants, ones with white background are not conserved, and those with green background have insufficient information to determine whether pathway position is conserved in land plants. Arrows and blunt‐ended arrows indicate positive and negative regulations, respectively. (a) In Marchantia, MpKAN regulates MpDELLA expression, which in turn negatively regulates MpPIF activity. Under high FR, MpPHY enters the nucleus and positively regulates MpPIF, which promotes the transition to sexual reproduction via gametangium initiation. (b) In Arabidopsis, phytochromeB (phyB) is nuclear localised under high R : FR, where it negatively regulates PIF activity via (1) protein–protein interactions resulting in degradation via the ubiquitin–proteasome system; and (2) reduced GA levels, which frees the DELLA transcription factor AtRGA to repress AtPIF4 and AtPIF5 activity. Under low R : FR (shade), phyB is located in the cytoplasm, resulting in increased PIF activity. PIF and KAN TFs act antagonistically to regulate expression of AtTAA1 expression, which in turn promotes hypocotyl elongation. Theoretical pathways are formulated based on results of this paper and previous work (Casal, ; Merelo et al., ; Inoue et al., ; Hernández‐García et al., 2021b). Question mark indicates partially supported pathway step where only AtKAN1 negatively regulates AtRGA (Merelo et al., 2013).