National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

HASEBE, Mitsuyasu

Assistant Professor:

ISHIKAWA, Masaki
SEGAMI, Shoji
Specially Appointed Assistant Professor:
MANO, Hiroaki
KOSETSU, Ken
Technical Staff:
OHI, Shoko
Postdoctoral Fellow:
NARUKAWA, Hideki
PALFALVI, Gergo
SOKENDAI Graduate Student: HORIUCHI, Yuta
DE VILLIERS, Ruan
CHEN, Peng
UEDA, Masamichi
Visiting Graduate Student: TANASE, Kuniaki
Visiting Undergraduate: MATSUDA, Riku
Technical Assistant:
AOKI, Etsuko
FUKADA, Hatsumi
HIRAMATSU, MikaKAJIKAWA, Ikumi
MATSUZAKI, Yoko
MASUOKA, Tomoko
NISHI, TayoZHANG, Liechi
OGAWA, Yuko
NAGATA, Kyoko
SAITO, Yuki
ASAOKA, Mayumi
BUSHRA, SamiraAdmin Support Staff:
KOJIMA, Yoko
HASEBE, Yuki

I. Evolution of Complex Adaptive Characters

The theory of natural selection and the neutral theory ofmolecular evolution are fundamental concepts in evolutionary biology. However, even with such theories, there still
remain unexplained phenomena, one of which is the evolution of complexity. It is difficult to explain the mechanisms
needed to evolve complex adaptive traits, because such traits
comprise plural components and become adaptive only when
all components are gathered together. However, based on
evolutionary theories, each component should evolve one by
one according to the accumulation of mutations.
To understand the molecular and cellular bases of adaptive
traits, we study the molecular and cellular mechanisms andevolution of (1) long distance signaling in plants, (2) reprogramming from differentiated cells to stem cells, and (3)development and morphology in land plants.

II. M olecular mechanisms and evolution oflong distance signaling in plants

Plants lack blood flow and nerves, but have evolved uniquelong-distance intercellular signaling mechanisms. Signals
using plant hormones, peptides, proteins, and slow calcium
waves have been well elucidated; however, the molecularmechanism of long-range, rapid, intercellular signaling byaction potentials with fast calcium waves, which evolved inparallel to similar signaling mechanisms in animals, remains
largely unknown. Rapid transmission of action potentials
has been reported in specific tissues of the sensitive plant
Mimosa pudica, the Venus flytrap Dionaea muscipula, andthe sundew Drosera rotundifolia. We use these species,
as well as Arabidopsis thaliana, to study the molecularmechanisms and evolution of action potential generation andtransmission. We have obtained genome sequences for thesespecies (Palfalvi et al. 2020 Curr. Biol. for Dionaea andunpublished for Mimosa pudica and Drosera rotundifolia)
and established techniques for transformation (Mano et al.
2014 PLoS ONE for Mimosa pudica; Suda et al. 2020 Nat.
Plants for Dionaea; unpublished for Drosera rotundifolia).
We intend to screen for genetic factors responsible for fast
intracellular and intercellular electrical signaling by action
potentials, analyze their molecular characteristics such as
effects on ion permeability and intracellular localization, andperform genetic gain- and loss-of-function experiments. This
will allow us to understand the general mechanisms of action
potential transmission in plants and the evolutionary processthat resulted in diversity of transmission velocity, which isadaptive in the three plants.

II-1. Molecular mechanisms of long distancesignaling of mechano-stimuli in thesensitive plant Mimosa pudica

The sensitive plant Mimosa pudica folds it leaves within acouple of seconds in response to touch. These rapid movements are mediated by specialized motor organs, pulvini,
which locate at the base of each compound leaves, pinnae,and small leaflets. When getting stimulated, motor cells in
one side of the pulvinus (extensor half) shrink while cells in
the opposite side (flexor half) do not shrink, consequently
leading to the unidirectional bending of the pulvinus. Because47National Institute for Basic Biology

Evolutionary Biology and Biodiversity

a pulvinus contains thousands of motor cells, achievementof the rapid movement at a whole pulvini requires not only
the rapid shrinkage of individual motor cells but also rapid
synchronization mechanisms among them. The molecularmechanisms underlying these rapid shrinkage and rapid cellcell communication still remain open questions.
To elucidate the molecular mechanisms for rapid leaf
movements, we have developed an efficient transformation
technique in M. pudica, and then established in planta live
imaging of Ca2+ with sensor fluorescent proteins and CRISPR/
Cas9-mediated gene knockout. To obtain candidate genes
responsible for rapid movements, we compared gene expression profiles between pulvini and non-motor organs, andbetween extensor and flexor halves of pulvini. As a result, wegot a number of extensor-enriched genes. After the systematic
knockout experiments of these candidate genes, mutants of
two channel genes, one gene encoding a membrane-associatedprotein with uncharacterized functions, and one transcription
factor showed deficiencies in touch-induced rapid movements.
Currently we are working on the detailed analysis on functionsof those genes and mutans by using transgenic techniques andelectrophysiological studies employing a heterologous expression system in Xenopus oocytes.
Rapid movements in M .pudica take only 1 to 2 seconds forthe whole process and our preliminary observations suggested
that their initial events like Ca2+ increase occurred within 100
ms or less. To achieve higher time resolution for our fluorescent
live imaging, we introduced a high-speed and high-sensitivity
camera system, and developed a method to analyze spatiotemporal propagation of Ca2+ signals robustly. We found that in
the tertiary pulvinus, a mechanical stimulus applied at the centerevokes a rapid concentric propagation of Ca2+ wave with 1.5
cm/s velocity, and it only took 30 ms to reach to the outermost
edge of excitable motor cells in the extensor half (Figure 1).
These analytical methods will facilitate future studies using
mutant lines described above. This study was mainly performed
by Dr. Hiroaki Mano with the support by technical staff.

elicited at the head by the stimulus and transmits through thestalk cells to the basal cells. The advantage of D. rotundifolia
to study transmission mechanisms of action potential is in thesimple structure of the tentacle, which is feasible for electrophysiology and bio-imaging. We succeeded to cultivate D.
rotundifolia under aseptic conditions with proper reactions
to the stimuli, and furthermore, to transform D. rotundifolia
with the green fluorescent protein gene. We improved thetransformation efficiencies with optimizing nutrient conditions in the medium.
In this fiscal year, nuclear genome of D. rotundifolia
was sequenced with long-read sequencing with Nanopore
sequencer and linked-read sequencing with TELL-seq library
and Illumina sequencer in 50 times coverage. To look forcandidate genes to function in the generation and transmission of action potential, we compared mRNA profiles
between different tissues in tentacles and between tentacles
and laminas. Loss-of-function experiments with CRISPR/
Cas9 mediated gene knockout are ongoing.
Ca2+ sensor RGECO expressed transformants were establishedto investigate the relationship between rapid calcium wave andaction potential transmission. Mechanical stimulation to thetentacle head produced a fast Ca2+ wave with similar speed tothe action potential, which was transmitted to the basal cells, but
not to laminar cells. We further investigate the molecular andcellular mechanisms with other transgenic lines.
The Venus flytrap is another carnivorous plant, whose leaf
blade bends to capture prey with two successive mechanicalstimuli applied to the sensory hairs on the surface. The mechanical stimulus is sensed at the middle cells of the hair, in which
action potential is generated to transmit to whole laminar cells.
As done in D. rotundifolia, we looked for candidate genes withcomparative transcriptomics, and gene knockout experiments
are ongoing. This study was mainly conducted by Dr. Shoji
Segami, Ms. Shoko Ooi, Mr. Peng Chen, Mr. Kuniaki Tanase,and Mr. Riku Matsuda with a the support of technical staff.
This work is collaboration work with Drs. Hiraku Suda andMasatsugu Toyota in Saitama University.

Figure 1. Fluorescence images of a red Ca2+ sensor R-GECO in a M.
pudica tertiary pulvinus after stimulated with a needle (100 fps). Right
image shows how the wave front propagates. 4 colors are repeatedly used
(R-B-Y-G) to discriminate each 10 ms frame.

II-2. Molecular mechanisms and evolution of
the generation and transmission of action
potential in the sundew Drosera rotundifolia
and the Venus flytrap Dionaea muscipula
Drosera rotundifolia is a carnivorous plant that captures
prey for nutrients by its tentacles, multicellular hairs on theleaf with sticky digestive fluid at their heads. Each tentacle
senses mechano- and chemical-stimuli at its head, and cells
in a tentacle base bend to bring the captured prey to thecenter of the trap leaf. In this process, action potential is48

Figure 2. Fluorescence images of a RGECO Drosera rotundifolia tentacle
after the tip was stimulated with a needle (red arrow in 0 sec), showingchanges of cytosolic calcium ion amounts. White arrows indicate frontsof calcium waves.

III. Molecular mechanisms of reprogrammingfrom differentiated cells to stem cells bySTEMIN

Stem cells self-renew and give rise to differentiated cellsto form tissues or organs. Differentiated cells in land plants
can be readily reprogrammed into stem cells during postembryonic development and regeneration. Thus, land plants
have an intrinsic ability to initiate a new genetic regulatory
network, despite the existence of epigenetic memory in differentiated cells. The underlying molecular mechanism andthe changes in the network due to mutation in its components
contribute to the environmental adaptability of land plants
to survive under harsh environments and to the evolution ofcomplex adaptive traits.
To understand the molecular basis of reprogramming, wehave used the moss Physcomitrium patens, in which leaf
cells can be reprogrammed into chloronema apical stem
cells by wounding. During this process, the expressionof STEMIN1 gene, which encodes AP2/ERF transcription
factor, is activated at leaf cells facing the cut. Deletion ofSTEMIN1 and its two homologs retards reprogramming,
while ectopic STEMIN1 induction changes differentiated cells into chloronema apical stem cells. We also foundthat transient DNA damage induces reprogramming ofleaf cells into stem cells, which requires the expression ofSTEMIN genes. Thus, STEMIN genes play a role in both thewounding- and the DNA damage-induced cellular reprogramming (Figure 3).
We found that ectopic induction of STEMIN1 in leaf cells
decreases a repressive chromatin mark, histone H3 lysine
27 trimethylation (H3K27me3), on its direct target genes.
To elucidate the mechanism regulating the local H3K27me3
reprogramming, we screened for STEMIN1-interacting
proteins and found that some of the components have thepotential to function in the histone modification. Thesesuggest that STEMIN1 could have a role as a platform torecruit components needed for local H3K27me3 reprogramming at the specific loci of the genome. This study wasmainly performed by Dr. Masaki Ishikawa and Mr. Ruan De
Villiers with the support of the technical staff.
Genotoxicregents

Heavy metals

IV. Evolution of Development and morphology
in land plants

Expansion of individual cells contributes to the morphogenesis of multicellular organisms, especially in land plants,
in which cell migration is restricted by the rigid cell wall.
Cell walls are mainly composed of the polysaccharides andthe outer lipid cuticle layer. Previous reports showed thatmechanisms acting on the cell wall polysaccharides, suchas cellulose and pectin, regulate cell expansion. However,
how other factors regulate cell expansion is not clear. We
found that an ATP-binding cassette (ABC) B family transporter, PpABCB14, localized on the plasma membrane of
the expanding region of gametophore initial cells and theleaf cells. Mr. Liechi Zhang and Dr. Masaki Ishikawa mainly
investigate the molecular mechanisms with support of thetechnical staff.

Publication List:〔Original papers〕

L., Inoue, N., Ishikawa, M., Gotoh, E., Teh, O.-K., Higa, T.,• Bao,Morimoto, T., Ginanjar, E.F., Harashima, H., Noda, N., Watahiki, M.,••••

High saltetc.

DNA damageWounding•DNA repair
ATR
PpCSP

STEMINPpWOX13L

ATG

Hiwatashi, Y., Sekine, M., Hasebe, M., Wada, M., and Fujita, T. (2022).
A PSTAIRE-type cyclin-dependent kinase controls light responses inland plants. Sci. Adv. 8, eabk2116. DOI: 10.1126/sciadv.abk2116
Kamada, T., Otomo, K., Murata, T., Nakata, K., Hiruma, S., Uehara,R., Hasebe, M., and Nemoto, T. (2022). Low-invasive 5D visualizationof mitotic progression by two-photon excitation spinning-disk confocal
microscopy. Sci. Rep. 12, 809. DOI: 10.1038/s41598-021-04543-7
Kanne, J. V, Ishikawa, M., Bressendorff, S., Ansbol, J., Hasebe, M.,
Rodriguez, E., and Petersen, M. (2021). Overexpression of ATG8/LC3
enhances wound-induced somatic reprogramming in Physcomitrium
patens. Autophagy. 18, 1463-1466.DOI: 10.1080/15548627.2021.1975913
Maeda, T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y.,Nagai, Y., Toyoda, A., Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N.,Nishiyama, T., Hasebe, M., Maruyama, T., Minagawa, J., Obokata, J.,
and Shigenobu, S. (2021). Chloroplast acquisition without the genetransfer in kleptoplastic sea slugs, Plakobranchus ocellatus. eLife 10,e60176. DOI: 10.7554/eLife.60176
Plant Cell Atlas Consortium, Jha, S.G., Borowsky, A.T., Cole, B.J.,
Fahlgren, N., Farmer, A., Huang, S.-S.C., Karia, P., Libault, M.,Provart, N.J., Rice, S.L., Saura-Sanchez, M., Agarwal, P., Ahkami,A.H., Anderton, C.R., Briggs, S.P., Brophy, J.A., Denolf, P., Di
Costanzo, L.F., Exposito-Alonso, M., Giacomello, S., Gomez-Cano,F., Kaufmann, K., Ko, D.K., Kumar, S., Malkovskiy V, A., Nakayama,N., Obata, T., Otegui, M.S., Palfalvi, G., Quezada-Rodriguez, E.H.,
Singh, R., Uhrig, R.G., Waese, J., Van Wijk, K., Wright,R.C., Ehrhardt,D.W., Birnbaum, and K.D., Rhee, S.Y. (2021). Science Forum: Vision,challenges and opportunities for a Plant Cell Atlas. eLife 10, e66877.
DOI: 10.7554/eLife.66877
Umeda, M., Ikeuchi, M., Ishikawa, M., Ito, T., Nishihama, R., Kyozuka,J., Torii, K.U., Satake, A., Goshima, G., and Sakakibara, H. (2021).
Plant stem cell research is uncovering the secrets of longevity andpersistent growth. Plant J. 106, 326–335. DOI: 10.1111/tpj.15184

andorCYCD;1Autophagy
EXPB4

H3K27me3

CDKA
orandCell cycle
reentry〔Review article〕

M., and Hasebe, M. (2022). Molecular mechanisms of• Ishikawa,reprogramming of differentiated cells into stem cells in the moss
Physcomitrium patens. Curr. Opin. Plant Biol. 65, 102123. DOI:
10.1016/j.pbi.2021.102123

Tip Growth

Figure 3. A working hypothesis of a genetic regulatory network of
cellular reprogramming in Physcomitrium patens.

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