DIVISION OF PLANT ENVIRONMENTAL RESPONSESProfessor

MORITA, Miyo T.

Assistant Professor:

NISHIMURA, Takeshi
SHIKATA, Hiromasa
Specially Appointed Assistant Professor:
KAWAMOTO, Nozomi
Research Staff:
TAKASE, Wakana
Technical Assistant:
HAMADA, Mayako
YAMADA, Yuka
SOMA, Yoriko
INOUE, Fumiko
MOTOMURA, Hiroe
Admin Support Staff:
KOJIMA, Yoko

physical signal trigger for biochemical signal transduction,
which in turn leads to the regulation of the polar auxin transport necessary to change the direction that a given plant isgrowing. We are investigating the detailed molecular mechanism of gravity sensing and signaling by using the model
plant Arabidopsis thaliana.
LAZY1 family genes have been shown to be involved in gravitropic responses in a variety of plants. LAZY1-LIKE(LZY)2,LZY3, and LZY4 are involved in root gravitropism ofArabidopsis. Previously, we have found that LZY3-mCherry
is polarly localized on the basal side of the plasma membranein the columella cells (root statocytes) in response to inclination. This suggests the close relationship between intracellular localization of LZY and gravity sensing. To elucidate
the gravity sensing mechanism, we are analyzing regions
responsible for intracellular localization of LZY3-mCherryand LZY4-mSacarlet by introducing mutations. A number oftransgenic lines harbouring mutated LZY-FP driven by theirown promoters in mutant background are under construction.

II. Determination mechanism of gravitropic
setpoint angle

Plant organs have the ability to sense various vectorial
stimuli such as light, humidity, touch and gravity as well
as reorient their growth direction so as to be in a suitable
position to survive and acclimate to their environment. Our
aim is to understand the molecular mechanism of environmental responses as they pertain to gravity in the main.

Visual overview of this lab’s work.

I. M olecular mechanisms of gravity sensing
and signaling in gravitropism

Growth angles affected by gravity are known as the gravitropic setpoint angles (GSA). Many gravitropic mutants show
abnormal GSA in lateral branches; meaning they produce
wider growth angle phenotypes due to the likelihood of
reduced gravitropism. We are trying to understand how roots’and shoots’ lateral branches maintain inclined growth angles
with respect to gravity.
It has been proposed that the GSA is determined by two
antagonistic growth components, gravitropism and antigravitropic offset (AGO). Currently, we are characterizing
Arabidopsis TAC1 (TILLER ANGLE CONTROL 1) gene asa candidate to control AGO. The TAC1 gene was originallyidentified by quantitative trait locus analysis in rice tiller
angle control (Yu et al., 2007). Later, TAC1 gene was identified in Peach tree (Prunus persica) as a causal gene of piller
phenotype (Dardick et al., 2013). In our lab, TAC1 gene
was found as a down regulated gene in agravitropic mutants
eal1 and sgr1 which lack an endodermis, a gravity sensing
tissue in shoot in Arabidopsis (Taniguchi et al., 2017). InArabidopsis, tac1 mutant has a compact shoot architecture
due to the upward lateral branch growth angles (Figure 1).
TAC1 encodes a protein with unknown function, but has asimilarity with LAZY1 family protein. However, while LZY
family proteins interact with RLD family proteins throughtheir conserved C-terminus in LAZY1 family protein (CCL)
domain and regulate gravitropism (Furutani et al., 2020),
TAC1 does not have a CCL domain, thereby TAC1 does notinteract with RLD family protein.
As well as characterizing TAC1 gene, currently, we are also
trying to isolate suppressor mutants of lzy1 lzy2 lzy3 by aforward genetic screening.

Gravitropism is a major determinant in directing plant organ
growth angles. In gravity sensing cells (statocytes), plastids
accumulating starch in high-densities relocate toward thedirection of gravity. Amyloplast relocation serves as a85

National Institute for Basic Biology

Environmental Biology

Figure 1. Growth angle phenotype of shoot lateral branches. Overallshoot architecture in wild type (A) and tac1 (B). Distribution of growth
direction of lateral branches in wild type (C) and tac1 (D). Growth angles
were plotted on histogram and distributions were evaluated in boxplot
from wild type (E) and tac1 (F).

III. F unctional analysis of RLD (RCC1-likedomain) proteins

RLD proteins were isolated as interactors of LZY. RLDs
function in gravitropism and the GSA control of roots. We
have also shown that RLD1, RLD2, RLD3, and RLD4 redundantly function in plant development and morphogenesis, themorphological abnormality gradually becomes more severe
by multiplying rld mutations (Figure 3). Indeed, rld1;2;3;4
quadruple mutants displayed severe embryonic development
defects due to a reduced amount and abnormal localization of PIN proteins (auxin efflux carriers) in the plasma
membrane. It had been reported that the RLD1 fragment
containing RCC1-like domain show guanine nucleotideexchange activity in Rab8 and Rab11. Thus, it’s likely thatRLD proteins regulate auxin transport through regulating
membrane trafficking during root gravitropism and development.
In columella cells, RLD1 is polarly recruited to the plasma
membrane by interacting with LZY3 and regulates the direction of auxin transport. The interaction between RLD1 andLZY3 occurs through the binding of the BRX domain ofRLD1 to the CCL domain of LZY3.
To understand further function of RLDs, we made attempt86

to identify the proteins that interact with RLD using yeast
two-hybrid screening. As a result, we found several proteinsas candidates for RLD interactor in addition to LZYs.
Among them, we further analyzed the polarity protein BASL(BREAKING OF ASYMMETRY IN THE STOMATALLINEAGE) which controls stomatal asymmetric cell division
as a collaboration with Dr. Dong and colleagues (Wang etal., 2021). Further analyses using yeast two-hybrid system
revealed that BASL interact with the BRX domain of RLD.
Developmental defects of rld1;2;3;4 mutant phenocopy
those of the severe allele of gnome mutants as previously
reported (Furutani et al., 2020). GNOM, a guanine nucleotide exchange factor for ARF (ADP ribosylation factor),
plays important roles in membrane trafficking. Dong andcolleagues found that RLD physically interacts with GNOM
in vitro and in vivo, and that GNOM and RLDs are requiredfor BASL polarization. Interestingly, we found GNOM in
the immunoprecipitates with LZY proteins (Furutani et al.,2020), implying that RLD-GNOM could be involved in LZY
function in the gravity signaling.
To investigate the importance of the BRX domain forRLD function, we examined whether RLD3p:RLD3∆BRXmClover3 could complement the morphological phenotype of rld2;3;4. Unexpectedly, RLD3∆BRX-mClover3
recovered the morphological abnormality of rld2;3;4(Figure 2). Interestingly, defects in gravity response
including GSA control and gravitropism were observedin RLD3p:RLD3∆BRX-mClover3/rld2;3;4. Both of these
indicate that RLD3 lacking the BRX domain retains thefunction for morphogenesis but not for gravity responsein shoot. Moreover, when RLD3 was expressed under
the control of the endodermis-specific ADF9 promoter inRLD3p:RLD3∆BRX-mClover3/rld2;3;4, the gravity response
was significantly restored, thus suggesting that RLD is
involved in gravity signaling in the endodermal cells inshoot gravitropism in a manner similar to LZY. These results
suggest that RLD function in several physiological events
including gravitropism, morphogenesis in BRX-dependentand -independent manner.

Figure 2. A. Phenotype of rld multiple mutants in mature plants. B. The
growth angle of lateral shoot in Wild Type (a), RLD3p:RLD3∆BRXmClover3/rld2;3;4 (b), ADF9p:RLD3/RLD3p:RLD3∆BRX-mClover3/
rld2;3;4 (c).

IV. Optogenetic approaches in plant biology
Optogenetics is a powerful, biological technique to manipulate the activity and localization of proteins in living cells,
thereby understanding their function. Many optogenetic tools
have been developed for the past three decades, however
there are few applications in plant biology because of bio-

logical and technical limitations; 1) plants use light forphotosynthesis or as their development cues and 2) most of
the tools are developed from plant light-sensitive proteins, inwhich light stimulating the tools may affect plant growth anddevelopment in an undesirable manner. Even so, optogenetic
approaches are worth introducing into plant biology because
they can control proteins of interest with a higher spatiotemporal resolution than those of conventional techniques likegene-inducible system and protein-uncaging with chemicals. We have been trying to optically control plant protein
kinases, particularly belonging to AGC kinase family, which
regulate fundamental cellular processes in plant growth anddevelopment, such as transport of plant hormone auxin andcell growth. We chose a strategy to control the kinase activity
directly by light, which can be controllable at the subcellularlevel. To carry out it, we fused light-responsive proteins/
modules to the kinases and analyzed their responsibility tolight. One of successful tools possess a blue light-sensitive
LOV2 domain from plant photoreceptor phototropin and its
interacting motif that we identified to negatively regulate
AGC kinase activity by blue light. By using this tool, wesucceeded to develop a light-sensitive kinase that directly
regulates the auxin transporter PINs, which is proven at leastby a heterogenous expression system with Xenopus oocytes.
It is expected that this tool will be a breakthrough techniquein plant biology to dissect lots of biological events mediatedby auxin (Figure 3).

Publication List:〔Original paper〕

R., Uchida, N., Tameshige, T., Kawamoto, N., Hotokezaka,• Fujihara,Y., Higaki, T., Simon, R., Torii, K.U., Tasaka, M., and Aida, M. (2021).

••

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2gene encoding a signaling peptide promotes cotyledon growth during
Arabidopsis thaliana embryogenesis. Plant Biotechnol. 38, 317–322.
DOI: 10.5511/plantbiotechnology.21.0508a
Kunita, I., Morita, M.T., Toda, M., and Higaki, T. (2021). A threedimensional scanning system for digital archiving and quantitativeevaluation of Arabidopsis plant architectures. Plant Cell Physiol. 62,
1975–1982. DOI: 10.1093/pcp/pcab068
Wang, L., Li, D., Yang, K., Guo, X., Bian, C., Nishimura, T., Le, J.,
Morita, M.T., Bergmann, D.C., and Dong, J. (2022). Connectedfunction of PRAF/RLD and GNOM in membrane trafficking controlsintrinsic cell polarity in plants. Nat. Commun. 13, 7. DOI: 10.1038/
s41467-021-27748-w〔Review article〕

M., and Morita, M.T. (2021). LAZY1-LIKE-mediated gravity• Furutani,
signaling pathway in root gravitropic set-point angle control. Plant
Physiol. 187, 1087–1095. DOI: 10.1093/plphys/kiab219

Figure 3. Schematic illustration of the light-sensitive AGC kinase. In
the dark, the chimeric AGC kinase is active to phosphorylate its target
proteins such as auxin transporter PINs (right). PINs are activated by thephosphorylation for efflux transport of indole-3-acetic acid (IAA). Uponblue light irradiation, the kinase activity can be reduced due to the blue
light-induced conformational change (left).

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