Cytokinin signaling coordinates development of diverse organs in Marchantia polymorpha

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(1)1. Short Communications (500–1000 words). 2. Title page. 3 4. Title: Cytokinin signaling coordinates development of diverse organs in Marchantia polymorpha. 5 6. Corresponding author: M. Umeda; Graduate School of Science and Technology, Nara Institute of. 7. Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0192, Japan; Tel. +81-743-72-5592;. 8. Fax +81-743-72-5599; E-mail: [email protected]. 9 10. 1 black and white figure, 1 color figure.. 11. 1.

(2) 12. Title: Cytokinin signaling coordinates development of diverse organs in Marchantia polymorpha. 13 14. Shiori S. Aki1, Ryuichi Nishihama2, Takayuki Kohchi2, Masaaki Umeda1,*. 15 16. 1. 17. 8916-5, Ikoma, Nara 630-0192, Japan. 18. 2. Graduate School of Science and Technology, Nara Institute of Science and Technology, Takayama Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. 19 20. *Corresponding author: E-mail, [email protected]; Fax, +81-743-72-5599. 21 22. Abbreviations: RR, response regulator; CKX, cytokinin oxidase.. 23. 2.

(3) 24. Abstract. 25. Cytokinins play an essential role in plant growth and development. A recent study showed that the. 26. cytokinin signaling pathway was conserved in the liverwort Marchantia polymorpha, and that it. 27. controlled gemma cup and rhizoid formation during thallus development. Here we show that the. 28. type-B response regulator, MpRRB, is mainly localized in the nucleus. Moreover, observations of. 29. thalli revealed that the distribution of air pores and the shape of the thallus margin are impaired in. 30. cytokinin-deficient lines and those defective in cytokinin signaling. This suggests that cytokinins. 31. regulate cell division and/or differentiation of precursor cells derived from the apical cell, thereby. 32. coordinating development of various organs produced on the thallus.. 33 34. Keywords. 35. Cytokinin, Marchantia polymorpha, organogenesis, response regulator. 36. 3.

(4) 37. The phytohormone cytokinin is involved in a broad range of physiological events, such as cell. 38. division, organ growth and senescence.1 Type-B response regulators (RRBs) are transcription factors. 39. that control cytokinin-responsive genes downstream of the two-component signaling pathway, which. 40. is mediated by CHASE domain-containing histidine kinase receptors and histidine-containing. 41. phosphotransfer proteins. Type-A response regulators (RRAs), one of the targets of RRBs, repress. 42. cytokinin signaling, thereby attenuating the response.2, 3 Genetic analyses using Arabidopsis mutants. 43. have revealed that both types of RRs play an essential role in various developmental processes,1 but. 44. how they precisely coordinate downstream events remains largely unknown.. 45. The liverwort Marchantia polymorpha is a model basal land plant.4, 5 The organization of. 46. the thalloid body derives from highly regulated division of the apical cell and its descendants at the. 47. thallus tip, which is called the notch. Arabidopsis possesses 10 and 11 genes encoding RRA and. 48. RRB, respectively. However, M. polymorpha only has one gene for each of RRA (MpRRA) and. 49. RRB (MpRRB). 6, 7 We recently reported that promoter activity of MpRRB was observed at the notch. 50. of young thalli and that transgenic plants defective in cytokinin signaling, e.g. Mprrb knockout lines,. 51. formed less or no gemma cup and more rhizoids than wild-type plants.7 Transgenic lines. 52. overexpressing cytokinin oxidase (CKX), which inactivates cytokinins,8 had less or no gemmiparous. 53. cells near the apical cell.7 These findings indicate that cytokinins participate in the early process of. 54. gemma cup and rhizoid formation. However, the involvement of cytokinin signaling in generating. 55. other organs or tissues has yet to be uncovered.. 56. In Arabidopsis, RRBs are localized in the nucleus to exert transcriptional activity.9, 10 To. 57. examine the subcellular localization of MpRRB, we fused a genomic fragment containing its. 58. promoter and coding regions to the gene for the fluorescent protein Citrine, and expressed the. 59. MpRRB-Citrine fusion protein in the Mprrb knockout line. As reported by Aki et al. (2019), 7 thalli. 60. with numerous rhizoids bent upward, and no gemma cup was formed in the Mprrb knockout line.. 4.

(5) 61. These phenotypes were suppressed in the two knockout lines expressing MpRRB-Citrine (#3 and #5). 62. (Figure 1A), indicating that MpRRB-Citrine is functional. To observe the localization of. 63. MpRRB-Citrine, gemmae were cultured for five days and fixed in PBS containing 4%. 64. paraformaldehyde at 4°C overnight, followed by clearing with the ClearSee solution for five days.11. 65. Citrine signals were detected at the apical notch (Figure 1B, upper), and a magnified view showed. 66. that a higher signal was observed in the nucleus while a significant level of fluorescence was also. 67. detected in the cytosol (Figure 1B, lower). We then transiently expressed MpRRB-Citrine in. 68. Arabidopsis protoplasts prepared from suspension cultured cells, and found that distinct Citrine. 69. signals were detected in the nucleus (Figure 1C). These results suggest that MpRRB is mainly. 70. localized in the nucleus, while it is possible that a small part of the protein is also present in the. 71. cytosol.. 72. To understand the role of cytokinins in organ development in more detail, we observed. 73. thalli of MpCKX2-overexpressing plants and knockout lines for MpRRA and MpRRB with scanning. 74. electron microscopy. Observations of the dorsal side of thalli revealed that air pores were uniformly. 75. dispersed in wild-type plants but were irregularly distributed in the MpCKX2-overexpressing line. 76. and the Mprrb knockout line; there was extensive variation in the distance between pores (Figure 2A,. 77. left). In the Mprrb knockout line, we found air pores without a circular shape (Figure 2B,. 78. arrowheads). We could not observe any differences in the distribution and the shape of air pores. 79. between wild-type plants and the Mprra knockout line, in which cytokinin signaling was activated. 80. (Figure 2A, left). Around the notch region, the MpCKX2-overexpressing and Mprrb knockout lines. 81. displayed ectopic serrations on the thallus margin, which was never observed in wild-type plants. 82. (Figure 2A, right). Ectopic serrations in MpRRB knockdown plants also have been described by. 83. Flores-Sandoval et al. (2016).12 We could not observe thallus margins of the Mprra knockout line,. 84. because they were buried in curled thalli (Figure 2A, right). Our results indicate that in addition to. 5.

(6) 85. gemma cup and rhizoid formation,7 cytokinin signaling controls the formation of air pores and. 86. thallus margins during thallus development.. 87. In M. polymorpha, all tissues in the thallus body derive from four merophytes (dorsal,. 88. ventral and two lateral merophytes) that are generated from the single apical cell at the notch.5. 89. Gemma cups originate from the dorsal merophyte, while rhizoids and air pores are produced from. 90. the ventral and dorsal sides of lateral merophytes, respectively. Although the origins of the thallus. 91. margin remain elusive, it is likely derived from lateral merophytes whose descendants undergo. 92. dorsiventral and/or coplanar cell division to form its smooth shape. While it remains unknown. 93. whether the observed defects in the MpCKX2-overexpressing and Mprrb knockout lines were due to. 94. impaired cell division or cell differentiation, our results suggest that cytokinin signaling regulates. 95. precursor cells derived from all merophytes, thereby enabling coordinated development of different. 96. organs during thallus growth. Further studies will be necessary to reveal the roles of cytokinins in. 97. cell division and/or differentiation during the early process of organ formation.. 98. 6.

(7) 99 100. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.. 101 102. Funding. 103. This work was supported by MEXT KAKENHI (17H06470 and 17H06477 to M.U., 18H04836 to. 104. R.N.) and JSPS KAKENHI (17H03965 to M.U., 16K07398 to R.N.).. 105. 7.

(8) 106. References. 107. 1. Kieber, J.J. and Schaller, G.E. (2014) Cytokinins. Arabidopsis Book 12: e0168.. 108. 2. To, J.P.C., Haberer, G., Ferreira, F.J., Deruère, J., Mason, M.G., Schaller, G.E., et al. (2004). 109. Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin. 110. signaling. Plant Cell 16: 658–671.. 111. 3. To, J.P.C., Deruère, J., Maxwell, B.B., Morris, V.F., Hutchison, C.E., Ferreira, F.J., et al. (2007). 112. Cytokinin regulates type-A Arabidopsis response regulator activity and protein stability via. 113. two-component phosphorelay. Plant Cell 19: 3901–3914.. 114 115 116 117. 4. Ishizaki, K., Nishihama, R., Yamato, K.T. and Kohchi, T. (2016) Molecular Genetic Tools and Techniques for Marchantia polymorpha Research. Plant Cell Physiol. 57: 262–270. 5. Shimamura, M. (2016) Marchantia polymorpha: Taxonomy, Phylogeny and Morphology of a Model System. Plant Cell Physiol. 57: 230–256.. 118. 6. Bowman, J.L., Kohchi, T., Yamato, K.T., Jenkins, J., Shu, S., Ishizaki, K., et al. (2017) Insights. 119. into land plant evolution garnered from the Marchantia polymorpha genome. Cell 171: 287–304.. 120. e15.. 121. 7. Aki, S.S., Mikami, T., Naramoto, S., Nishihama, R., Ishizaki, K., Kojima, M., Takebayashi, Y.,. 122. Sakakibara, H., Kyozuka, J., Kohchi, T., Umeda, M.. (2019) Cytokinin Signaling is Essential for. 123. Organ Formation in Marchantia polymorpha. Plant Cell Physiol. 60: 1842–1854.. 124. 8. Schmülling, T., Werner, T., Riefler, M., Krupkova, E., Bartrina, Y. and Manns, I. (2003). 125. Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis. 126. and other species. J. Plant Res. 116: 241–252.. 127 128 129. 9. Mason, M.G., Li, J., Mathews, D.E., Kieber, J.J., Schaller, G.E. (2005) Type-B response regulators display overlapping expression patterns in Arabidopsis. Plant Physiol. 135: 927–937. 10. Xie, M., Chen, H., Huang, L., O'Neil, R.C., Shokhirev, M.N., Ecker, J.R. (2018) A. 8.

(9) 130. B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot. 131. development. Nat Commun. 9: 1604–1616.. 132 133 134 135. 11. Kurihara, D., Mizuta, Y., Sato, Y. and Higashiyama, T. (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142: 4168–4179. 12. Flores-Sandoval, E., Dierschke, T., Fisher, T.J. and Bowman, J.L. (2016) Efficient and inducible use of artificial microRNAs in Marchantia polymorpha. Plant Cell Physiol. 57: 281–290.. 136. 13. Ishizaki, K., Nishihama, R., Ueda, M., Inoue, K., Ishida, S., Nishimura, Y., et al. (2015). 137. Development of gateway binary vector series with four different selection markers for the. 138. liverwort Marchantia polymorpha. PLoS One 10: e0138876.. 139. 9.

(10) 140. Figure Legends. 141. Figure 1. Subcellular localization of MpRRB. (A) Thalli of the Mprrb knockout line and the same. 142. line carrying proMpRRB:MpRRB-Citrine. The genomic fragment containing the promoter and the. 143. coding regions of MpRRB was introduced into the binary vector pMpGWB307,13 which was then. 144. transformed into the Mprrb knockout line having an F1 background generated by crossing Tak-1 and. 145. Tak-2. Thallus tips were cultured for 15 days. Arrowheads indicate gemma cups. Bars represent 1 cm.. 146. (B) Expression and protein localization of Citrine-fused MpRRB at the notch. Gemmae were. 147. cultured for five days, and harvested thalli were fixed and cleared. Images were obtained with the. 148. confocal microscope (FV1000, Olympus). The lower panels are enlarged images of the notch region.. 149. Fluorescence images of Citrine signals (left); brightfield images (middle); merged images (right).. 150. Arrowheads indicate apical notches. Bars represent 100 µm. (C) Protein localization of Citrine-fused. 151. MpRRB in Arabidopsis protoplasts. MpRRB-Citrine was expressed under the cauliflower mosaic. 152. virus 35S promoter in Arabidopsis protoplasts prepared from suspension cultured cells. Fluorescence. 153. image of Citrine signal (upper left); DAPI-stained nuclei (upper right); bright-field image (lower. 154. left); merged image (lower right). Bar represents 20 µm.. 155 156. Figure 2. Scanning electron micrographs of the MpCKX2-overexpressing line and the knockout. 157. lines of MpRRA and MpRRB. (A) Apical notch regions (left) and thallus margins (right). The. 158. MpCKX2-overexpressing line and the knockout lines of MpRRA and MpRRB are male and female. 159. lines, which have Tak-1 and F1 background, respectively. (B) Magnified images of the dorsal side of. 160. thalli. Thallus tips were cultured for 14 days and observed with a scanning electron microscope. 161. TM3000 (Hitachi High Technologies). Arrowheads indicate air pores without a circular shape. Bars. 162. represent 200 µm.. 10.

(11) A. proMpRRB:MpRRB-Citrine/Mprrbko WT (Tak-2). B. Figure 1. Mprrbko. #3. #5. C MpRRB-Citrine. DAPI. Bright-field. Merge.

(12) Mprrako Mprrbko WT (Tak-2). WT (Tak-1). WT (Tak-2). proMpEF1α: MpCKX2. Mprrbko. A B. Figure 2.

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