Marchantia MpRKD Regulates the Gametophyte-Sporophyte Transition by Keeping Egg Cells Quiescent in the Absence of Fertilization  Moritz Rövekamp, John L. Bowman, Ueli Grossniklaus  Current Biology  Volume 26, Issue 13, Pages 1782-1789 (July 2016) DOI: 10.1016/j.cub.2016.05.028 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Viridiplantae RKD Genes Form a Monophyletic Group Unrooted phylogenetic tree of the RWP-RK homologs of the Viridiplantae reconstructed with Bayesian inference from aligned amino acid sequences (Figure S1). Bayesian inference was calculated with ten million Markov Chain Monte Carlo (MCMC) generations. Posterior probability values above 0.9 are indicated by full circles, and empty circles denote posterior probability values above 0.7 of the respective nodes. Five clades were retrieved, four of which contained land plant sequences (denoted by colored arcs). Previous analyses used the only non-plant sequences from Dictyostelium to root the tree [19], but our analysis suggests these were acquired by the slime mold via horizontal gene transfer from a land plant. Thus, we named the clades after the first characterized genes in each as follows: RKD (RKD(A) of Chardin et al. [19]), MID (similar to RKD(C) of Chardin et al. [19]), NIN (NLP of Chardin et al. [19]), and an unnamed clade (red) lacking flowering plant genes. M. polymorpha homologs are in bold and their position is indicated with a star: Mapoly0022s0128 (MpRKD), Mapoly0083s0040 (MpNIN), Mapoly0014s044 (MpMID), and Mapoly0049s0118 (MpRWP). All RKD clade genes identified shared the conserved RKD and a second domain, to which Chardin et al. [19] referred to as motif 12, and related sequences can be found not only in genes of the RKD clade but also in other sequences indicated in blue. Likewise, sequences highlighted in red and green, respectively, also share conserved sequences outside the RWP-RK domain. Some of the S. moellendorfii and charophycean algal sequences are incomplete. Mp, Mapoly. See also Figures S1 and S2. Current Biology 2016 26, 1782-1789DOI: (10.1016/j.cub.2016.05.028) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Expression Pattern of MpRKD in Different Tissues (A–G and I) Plants expressing a construct where the native MpRKD promoter drives expression of the trpVNS fluorescent protein and (H and J) wild-type control plants. (A) Young thallus. Meristematic zones called apical notches are indicated by arrows. (B) Gemma cup indicated by an arrow. (C) Antheridiophore. (D) Archegoniophore. (E) Developing sporophyte, 10 days after fertilization. Contours are indicated in white. (F) Ruptured sporophyte, 16 days after fertilization. Contours and rupture are indicated in white with dotted and dashed lines, respectively; emerging in rows from the rupture are spores expressing trpVNS and elaters not expressing trpVNS. (G) Antheridia indicated by arrows. (H) Wild-type antheridia indicated by arrows. (I) Archegonia indicated by arrows. The egg cells show strong trpVNS expression. (J) Wild-type archegonia indicated by arrows. (K) RT-PCR for MpRKD and the control gene MpEF1α in different tissues after (I) 24 cycles, (II) 28 cycles, and (III) 33 cycles. Lanes with size marker are indicated in kilobases. Bands and ladders were all run on the same gel and assembled for this figure. Scale bars, 2 mm (A and C), 500 μm (B), 1 mm (D), 100 μm (E–H), and 75 μm (I and J). See also Figure S3. Current Biology 2016 26, 1782-1789DOI: (10.1016/j.cub.2016.05.028) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Gemma Cups Are Not Formed and Gemmae Grow More Slowly in amiRKD Transformants (A–F) Scanning electron micrographs of gemma cups in wild-type plants (A–C) and the missing gemma cups in amiRKD transformants (D–F). (G) The qRT-PCR on MpRKD comparing different tissue types of wild-type plants (white bars) and plants expressing a pMpEF1α::amiRKDMpmiR160 construct (gray bars). Significant differences are indicated according to Student’s t test and Wilcoxon rank-sum test (∗∗∗p < 0.001). Thallus and gametophore tissues were analyzed in two independent qRT-PCR reactions. See also Figure S4A and Table S1. (H) Growth rate of wild-type and amiRKD gemmae. Gemmae were cultured on plates containing half-strength Gamborg B5 medium, and plates were regularly scanned to measure the increase in surface area. The area is indicated in mm2, time in days after plating (DAP), asterisks indicate a significantly different growth rate within an interval (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001), and error bars indicate the 95% confidence interval. See the Supplemental Experimental Procedures for details and Table S2. Scale bars, 1 mm (A and D), 500 μm (B), 200 μm (C and E), and 100 μm (F). See also Figure S4. Current Biology 2016 26, 1782-1789DOI: (10.1016/j.cub.2016.05.028) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 MpRKD Is Required for the Establishment and/or Maintenance of Egg Cell Quiescence in the Absence of Fertilization Archegonia contours are indicated in red, and contours of egg cells and the descendants of dividing egg cells are indicated in black. (A) Mature archegonia in wild-type plants with clearly visible, large egg cells. (B–D) Archegonia in MpRKD knockdown lines, showing that the egg cell starts divisions before the archegonium has reached full maturity. (B) Mature archegonium in amiRNA transformant, with dividing cells clearly visible at the position of the egg cell. (C) Immature archegonium at early stage in amiRNA transformant (neck is still short and closed and a large cell at the base is clearly visible that is morphologically indistinguishable from wild-type egg cells). (D) Immature archegonium at a later stage in amiRNA transformant (neck is more elongated but still closed and the large cell at the base has started to divide in the absence of fertilization, while the archegonium still develops). (E and F) Growth of ectopic archegoniophores is observed in MpRKD knockdown lines at later stages. (G) Frontal section of z stack of a wild-type archegonium with colored egg cell surfaces. (H) Extracted surfaces of wild-type egg cells. (I) Frontal section of z stack of an amiRKD-transformed archegonium with colored dividing cells. (J) Extracted surfaces of dividing cells. (K and L) Frontal section of z stack of young sporophytes. (M and N) Extracted surfaces of young sporophytes. Sporophytes were at the beginning of the fourth round of division (K and M) or at a later stage with about 30 cells (L and N). Scale bars, 10 μm (A–D), 1 mm (E and F), 200 μm (G), 100 μm (H and K–M), and 50 μm (I, J, and N). See also Table S3 and Movie S1. Current Biology 2016 26, 1782-1789DOI: (10.1016/j.cub.2016.05.028) Copyright © 2016 Elsevier Ltd Terms and Conditions