470 Proc. Jpn. Acad., Ser. B 98 (2022) [Vol.

98,

Review
Functional genomics in plant abiotic stress responses and tolerance:
From gene discovery to complex regulatory networks
and their application in breeding

By Kazuo SHINOZAKI*1,† and Kazuko YAMAGUCHI-SHINOZAKI*2,*3

(Edited by Yoshinori OHSUMI, M.J.A.)

Abstract: Land plants have developed sophisticated systems to cope with severe stressful
environmental conditions during evolution. Plants have complex molecular systems to respond and
adapt to abiotic stress, including drought, cold, and heat stress. Since 1989, we have been working to
understand the complex molecular mechanisms of plant responses to severe environmental stress
conditions based on functional genomics approaches with Arabidopsis thaliana as a model plant. We
focused on the function of drought-inducible genes and the regulation of their stress-inducible
transcription, perception and cellular signal transduction of stress signals to describe plant stress
responses and adaptation at the molecular and cellular levels. We have identified key genes and
factors in the regulation of complex responses and tolerance of plants in response to dehydration and
temperature stresses. In this review article, we describe our 30-year experience in research and
development based on functional genomics to understand sophisticated systems in plant response
and adaptation to environmental stress conditions.

Keywords: drought and temperature stress, hormone, transcriptome, signal transduction,

long-distance signals, molecular breeding

thesis is prohibited by the reduction in gas exchange.

Introduction
Abscisic acid (ABA) is a key phytohormone that
Plants are sessile organisms and are able to not only prevents water loss by transpiration from
adapt to severe environmental conditions for growth, guard cells but also enables adaptation to drought
development and survival. During evolution in stress conditions. ABA accumulates in drought stress
stressful environmental conditions, land plants have conditions and quickly spreads intercellularly in
developed unique systems to propagate environ- whole tissues to induce plant stress responses and
mental stress signals from sensing tissues to appro- adaptation.3)–6) In addition to physiological research,
priate targets to achieve optimal growth mainte- molecular and cellular biology have contributed to
nance and tolerance to stress conditions.1),2) Land resolving the precise regulatory gene networks
plants respond and adapt to adverse environmental associated with complex abiotic stress responses
conditions, such as drought, high and low temper- and resistance in model plants such as Arabidopsis
atures, and high salinity. During drought stress, thaliana and rice in abiotic stress conditions.4),7),8)
plants close stomata to save water, and photosyn- When we started our laboratory at the RIKEN
Tsukuba Life Science Center in 1989, we focused our
*1 RIKEN Center for Sustainable Resource Science, work on molecular biological analyses of plant
Tsukuba, Ibaraki, Japan.
*2
environmental responses, especially drought stress,
Research Institute for Agricultural and Life Sciences,
Tokyo University of Agriculture, Tokyo, Japan. because water deficit conditions have a severe effect
*3 Graduate School of Agricultural and Life Sciences, The on plant growth. We selected Arabidopsis thaliana as
University of Tokyo, Tokyo, Japan. a model plant for this purpose because of its high
†
Correspondence should be addressed to: K. Shinozaki,
potential for genomics research.9) In the late 1980s,
RIKEN Center for Sustainable Resource Science, 3-1-1 Koyadai,
Tsukuba, Ibaraki 305-0074, Japan (e-mail: kazuo.shinozaki@ protein analyses revealed the accumulation of specific
riken.jp). proteins in response to low and high temperature and

doi: 10.2183/pjab.98.024
©2022 The Author(s). Published under the terms of the CC BY-
NC license https://creativecommons.org/licenses/by-nc/4.0/.
No. 8] Regulatory gene networks in plant abiotic stress responses 471

water deficiency. Accumulated proteins include late over, we have tried to apply basic knowledge
embryogenesis abundant (LEA) proteins and heat obtained with Arabidopsis for application to the
shock proteins, which function in protecting cells breeding of drought-tolerant crops.7),12)
from stress conditions.1) These observations led us In this review article, we mainly focus on our
to hypothesize stress-inducible expression of stress- functional genomics studies to understand complex
specific genes whose products function in stress plant responses to abiotic stress, especially drought
tolerance and response in complex environmental and temperature stresses, and regulatory systems
stresses.9) To understand the molecular and cellular for abiotic-stress tolerance.7),11),13),14) Recent studies
basis of plant responses to complex abiotic stress, on intertissue and interorgan signaling in plant stress
especially drought, cold, and heat stresses, we have responses and adaptation are also summarized
used reverse genetics and functional genomics ap- (Fig. 1B).15)–17) Moreover, the importance of quanti-
proaches to isolate many drought-inducible genes tative phenotyping analyses of plant growth in
with various functions and analyze their regulatory water-deficit conditions is discussed.17) The applica-
systems of gene expression in plant abiotic stress tion of basic knowledge on plant stress resistance for
responses.4),10) We focused our work on analyzing the molecular breeding of drought-tolerant crops is
transcriptional regulation in abiotic stress responses discussed to contribute to crop production in dry
and their associated signaling networks (Fig. 1A). areas during global warming.12) In this review, we
We discovered many important regulatory genes summarized our research strategies and accomplish-
involved in plant responses to drought, cold, and heat ments on molecular and cellular analyses of regu-
and analyzed gene expression and signal transduction latory systems of plant responses to environmental
in abiotic stress responses. We discovered ABA- abiotic stresses, such as drought, cold, and heat
independent regulatory systems in the plant response stresses.
to drought stress in addition to those that are ABA
Gene discovery in the plant environmental
dependent.10) We also analyzed ABA-dependent
stress response
regulatory processes of drought stress responses,
including transcriptional regulation, signal trans- Since 1989, we have cloned numerous genes
duction and intertissue communication.7),11) More- involved in the environmental stress response and

Fig. 1. Plant responses to drought stress: From regulatory gene networks in cellular responses to integrative interorgan signaling for
stress tolerance. A. Molecular genetics and functional genomics have elucidated the complex cellular processes of plant responses to
environmental stresses, including regulation of gene expression and cellular signal transduction. B. Plant hormones, including ABA
and peptides, function in intertissue and interorgan signal transduction for the integration of stress signals driving proper responses
throughout the entire plant. This figure is reprinted with modification from Ref. 17.
472 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

resistance acquisition, analyzed their gene structure stress-related genes. Transcriptional regulation plays
and function, and then analyzed the regulation of important roles in the acquisition of the environ-
gene expression in drought and low-temperature mental stress response and tolerance in plants.
conditions. First, we prepared cDNA libraries from
Functions of drought stress-inducible genes
Arabidopsis plants that were dehydrated and
screened cDNAs of the mRNAs that were upregu- The expression of many plant genes with various
lated in response to drought stress.18),19) We have functions is regulated by abiotic environmental
shown that many genes are induced by abiotic stresses such as drought, heat, and cold. Tran-
stress, such as drought and low temperature, and scriptome analysis using microarray technology is a
that many of these genes are involved in the powerful technique that has proven to be very useful
acquisition of stress tolerance. Later, we used micro- for the discovery of many stress-inducible genes
arrays to identify drought-upregulated genes in involved in stress response and tolerance.20),21)
Arabidopsis and other crops.20),21) Drought stress- Numerous genes that are induced by various abiotic
inducible genes at the transcriptional level include stresses have been identified using different micro-
not only genes regulated by the plant hormone ABA array systems.10),25)
but also genes induced by stress without ABA. ABA Genes induced during stress conditions are
is produced in water-deficit conditions and plays an thought to function not only in protecting cells from
important role in plant drought tolerance. Exogenous stress by the production of important metabolic
application of ABA also induces several genes that proteins but also in the regulation of genes for signal
respond to dehydration.22) Several reports have transduction in the stress response (Fig. 2). Thus,
described genes that are induced by dehydration these gene products are classified into two major
and cold stresses but that do not respond to groups with different functions.9),10) The first group
exogenous ABA treatment.22)–24) This suggests the includes proteins that probably function in stress
existence of ABA-independent, as well as ABA- tolerance, such as chaperones, late embryogenesis
dependent, signal transduction cascades between the abundant (LEA) proteins, osmotin, antifreeze pro-
initial stress signals and the expression of specific teins, key enzymes for osmolyte biosynthesis such as

Functional proteins Regulatory proteins

Water channel,
transporters Transcription factors
Detoxification (DREB2, AREB, MYC,
enzymes to reduce ROS MYB, bZIP, NAC
HB etc.)

Protection factors Protein kinases,

of macromolecules Drought phosphatases
(LEA proteins,
Phospholipid
chaperons)
metabolism

Key enzymes for ABA Regulation

osmolyte biosynthesis
(proline, sugars) Proteases
Fig. 2. Functions of drought-inducible genes in stress tolerance and responses. Thousands of drought-inducible genes have been
identified by transcript analyses. These gene products are classified into two major groups: functional proteins involved in cellular
stress tolerance and regulatory proteins involved in gene expression and signal transduction of stress responses. In addition to these
proteins, noncoding RNAs and small peptides have been reported to function in the regulation of stress responses. Names and
functions of genes for stress-inducible genes are summarized in Table 1.
No. 8] Regulatory gene networks in plant abiotic stress responses 473

proline and raffinose, water channel proteins, sugar cooperatively regulate various stress-inducible genes
and proline transporters, detoxification enzymes to and may constitute transcriptional gene networks.
reduce reactive oxgen spieces (ROS), enzymes for Functional analysis of these stress-inducible tran-
fatty acid metabolism, and proteinase inhibitors. scription factors should provide more information on
Overexpression of these stress-inducible genes that the complex regulatory gene networks that are
encode key enzymes for osmolyte biosynthesis, LEA involved in responses to drought, cold, and high-
proteins, and detoxification enzymes has been shown salinity stresses. The others were protein kinases,
to improve stress-tolerant phenotypes of transgenic protein phosphatases, enzymes involved in phospho-
plants.12),25) These results indicate that the gene lipid metabolism, and other signaling molecules.
products of the stress-inducible genes truly function Some of these stress-inducible regulatory genes
in stress tolerance in plants. have been overexpressed in transgenic plants to
The second group contained protein factors generate stress-tolerant phenotypes in transgenic
involved in the further regulation of signal trans- plants.2),7),12)
duction and gene expression that probably function Many noncoding RNA transcripts have also
in the stress response (Fig. 2). They include various been identified as stress-inducible genes. These
types of transcription factors, suggesting that various include microRNAs, antisense RNAs and long-non-
transcriptional regulatory mechanisms function in coding RNAs.26),27) Some of these noncoding RNAs
drought-, cold- or high salinity-stress signal trans- function in the regulation of stress responses. Stress-
duction pathways.9)–11) These stress-inducible tran- inducible expression of regulatory genes is a typical
scription factors include members of the DRE-bind- response and adaptation of plants against adverse
ing protein (DREB), ethylene-responsible element environmental stress conditions.
binding factor (ERF), zinc-finger, WRKY, MYB, Table 1 summarizes a list of names of genes and
basic helix-loop-helix (bHLH), basic-domain leucine factors that we discovered during our research on
zipper (bZIP), NAC, and homeodomain transcription plant responses to abiotic stress like drought, heat,
factor families.10) These transcription factors can and cold.

Table 1. Names of genes and factors involved in abiotic stress responses and tolerance

Gene name Functions Abiotic stress References

Stress-inducible genes involved in stress tolerance
LEA Late embryogenesis abundant Dry, Cold 10
RD29 Responsive to dehydration 29 Dry, Cold, ABA 10, 18
RD22 Responsive to dehydration 22 Dry, ABA
10, 22
Unidentified seed protein
ERD1 Early responsive to dehydration 1 Dry, ABA
10, 19, 42
Clp protease regulatory subunit
HSP Heat shock protein Heat, Dry 10, 13
GolS Galactinol synthase Dry, Cold, Heat 61–63
GAD GABA decarboxylase Dry, ABA 61

Transcription factors
DREB1/CBF DRE-binding protein 1/ Cold
10, 28, 30
C-repeat binding factor
DREB2 DRE-binding protein 2 Dry, Heat 10, 28, 34
AREB/ABF ABRE-binding protein TF ABA, Dry 10, 36, 37
AtMYC2 MYC related TF 2 ABA, Dry 10, 39, 40
AtMYB2 MYB related TF 2 ABA, Dry 10, 40
NAC/RD26 NAC TF Dry 10, 43, 44
ZF-HD Zn finger-homeodomain TF Dry 10, 45
HB6 Homeobox 6 TF ABA, Dry 10, 46
TCP13 TCP family 13 TF Dry 47
Continued on next page:
474 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

Continued:
bZIP60 bZIP TF in ER stress response ER 48
bZIP17/28 bZIP TF in ER stress response ER 49
TAF12b TATA binding factor 12b ER 50
HSFA1 Heat shock factor A1 Heat 13
CAMTA1 Calmodulin-binding TF Cold 14, 56, 57
RVE Reveille TF Cold 14, 57

ABA related enzymes and transporters

NCED 9-cis-epoxycarotenoid dioxygenase ABA synthesis 104–106
CYP707A P450 cytochrome oxidase ABA degradation 104–106
ABC ATP-binding cassette transporter ABA transport 110–114
NPF Nitrate/peptide transporter ABA transport 116–118
DTX Detoxification efflux carrier ABA transport 119

Signaling factors in stress responses

SnRK2 SNF-like protein kinase 2 ABA, Dry 11, 67, 68
PP2C Protein phosphatase 2C ABA, Dry 3, 5
PYR/PYL/RCAR START-type ABA receptor ABA 73, 74
Raf-like kinase MAPK kinase kinase (MAPKKK) ABA, Dry 81–83
VCS VARICOSE mRNA decapping Dry 11, 84
CK1 Casein kinase 1 Dry 55
CPK Ca2D-dependent protein kinase Dry, Cold, ABA 11
ATHK1/AHK1 Arabidopsis histidine kinase 1 Dry 87–89
MCA1 MID1 homolog of yeast Dry, Cold 92, 93
Ca2D-permeable stretch-activated channel
OSCA1 Hyperosmolarity induced Ca2D increase Dry 96–98
HPCA H2O2-induced Ca2D increase ROS 101
RBOHD Respiratory burst oxidase D ROS 101, 103
CLE25 Clavata3 related peptide 25 Dry 121
Genes and factors involved in abiotic stress responses and tolerance are listed in this table and discussed in the text.
Dry: drought stress; Cold: cold stress; Heat: heat stress; ABA: abscisic acid regulated; ER: endoplasmic reticulum; ROS: reactive
oxygen species; TF: transcription factor.

as bHLH transcription factors, DREB1A, DREB2A,

Transcriptional regulation of
AP2/ERF domain-containing protein RAP2, and
stress-inducible genes
growth factor-like protein.10) In the other group,
The expression patterns of genes induced by gene expression slowly and gradually increased after
abiotic stresses have been analyzed by RNA-blot stress treatment within 10 hours. Most of these genes
hybridization analyses and recently using micro- encode functional proteins such as LEA proteins,
arrays, RNA sequencing, and quantitative PCR. detoxification enzymes and enzymes for osmolyte
Through these transcriptome studies, thousands of synthesis. However, some of these stress-inducible
plant genes have been shown to be stress inducible. genes respond to ABA, whereas others do not. ABA-
Only 10% of drought-inducible genes were also deficient mutants were used to analyze drought-
induced by cold stress.21) The results also indicated inducible genes that respond to ABA. Many genes
broad variations in the timing of induction of these were induced by exogenous ABA treatment. How-
genes, and there are at least two groups showing ever, some genes were induced by cold or drought in
different expression profiles.17) In one group, gene ABA-deficient (aba) or ABA-insensitive (abi) mu-
expression was rapid and transient in response to tants. This means that there are not only ABA-
drought and cold stresses, reached a maximum dependent systems but also ABA-independent sys-
within several hours, and then decreased. Most of tems involved in the drought stress response
these genes encoded regulatory protein factors such (Fig. 3).10)
No. 8] Regulatory gene networks in plant abiotic stress responses 475

Fig. 3. Transcriptional regulatory gene network in plant abiotic stress responses, such as drought, heat, and cold stresses. ABA is a
major plant hormone associated with the drought stress response. There are ABA-dependent regulatory systems and ABA-
independent systems involved in the drought stress response. The bZIP transcription factors AREBs/ABFs function in ABA-
dependent regulation. In ABA-independent pathways, DREB2s encoding AP2 transcription factor function in drought-inducible
transcription. CK1 protein kinase is involved in the activation of DREB2s. In addition, NAC, ZF-HD, MYB, and MYC transcription
factors are involved in the drought stress response. In the heat stress response, HSFA2 transcription factors are major regulatory
factors and regulate downstream transcription factors, including DREB2s. In the cold stress response, DREB1s/CBFs are major
transcription factors in cold-inducible transcription. Cold-inducible induction of DREB1/CBF genes is regulated by 2 different
transcription factors: CAMTA TF in the rapid response and CCA1/LHY and RVE in circadian regulation. Names and functions
of genes for transcription factors (TFs; shown in circles) and signaling factors (SFs; shown in hexagons) are summarized in Table 1.
Cis-acting elements are shown as rectangles. Receptors and sensors have not been clearly identified and shown with dashed lines.

Transcription factors involved in ABA-depend- DRE is also found in the promoter regions of many
ent and ABA-independent gene expression in abiotic cold-inducible genes and is named C-repeat (CRT)
stress responses are summarized in Table 1. and low-temperature-responsive element, both con-
taining an A/GCCGAC motif that forms the core
ABA-independent transcription in response
of the DRE sequence and regulates cold-inducible
to drought and/or cold stress
promoters.29) The cDNAs encoding DRE/CRT bind-
We precisely analyzed the expression profiles ing proteins, C-repeat binding factor/DRE binding
of the Arabidopsis RD29A/COR78/LTI78 gene, the protein 1 (CBF/DREB1), and DREB2, have been
product of which protects cells from dehydration. isolated using yeast one-hybrid screening.30),31) These
The RD29A gene is induced by drought, cold, and proteins contained the conserved DNA-binding
ABA treatment. However, this gene is induced in aba domain found in the ethylene-responsive element
or abi mutants by both drought and cold stresses, binding factor (ERF) and Apetala2 (AP2) proteins.
which indicates that it is governed by both ABA- These proteins specifically bind to the DRE/CRT
dependent and -independent regulation in drought sequence and activate the transcription of genes
and cold conditions.22) Analysis of this RD29A driven by the DRE/CRT sequence (Fig. 3).
promoter has shown that a 9-bp conserved sequence, In Arabidopsis, three genes encoding DREB1/
TACCGACAT, named the dehydration responsive CBF lie in tandem on chromosome 4 in the following
element (DRE), is an essential cis-acting element order: DREB1B/CBF1, DREB1A/CBF3, and
for the regulation of RD29A induction in the ABA- DREB1C/CBF2. Expression of the DREB1/CBF
independent response to dehydration and cold.28) genes is induced by cold stress but not by dehydra-
476 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

tion or high-salinity stress. Three DREB1 proteins regulatory subunit responds to dehydration and high
are major transcription factors involved in cold- salinity before the accumulation of ABA. This
induced gene expression.30)–33) There are two DREB2 pattern of induction suggests that an ABA-inde-
proteins, DREB2A and DREB2B. Expression of the pendent pathway exists in the dehydration stress
DREB2 genes is induced by dehydration and high- response of Arabidopsis.42) Analysis of the ERD1
salinity stresses but not by cold stress.34) Both promoter revealed two novel cis-acting elements, a
DREB2A and DREB2B proteins function in drought 14-bp rps1-like region (CACTAAATTGTCAC) and
responses and are discussed in more detail in a later a CATGTG motif, which are necessary for the
section on roles of DREB2. induction of ERD1. Three NAC transcription factors,
including RD26, bind the c-acting element to
ABA-dependent transcription and its regulation
upregulate ERD1.43),44) Moreover, ZF-HD proteins
ABA plays an important role in the signal are necessary for the NAC-activated expression of
transduction of osmotic stress in plants. ABA- ERD1.45) This regulatory system is different from the
responsive element (ABRE: ACGTGG/TC) is a DREB/CBF system in the ABA-independent induc-
major cis-acting element that functions to regulate tion of stress genes.
ABA-responsive gene expression.10),35) In Arabidop- The homeobox protein HB6 functions as a
sis, two ABRE motifs are important for the negative regulator of the ABA response.46) HB6
regulation of ABA-responsive expression of the interacts with ABA-inducible promoters with the
RD29B gene encoding a LEA-like protein. The basic CAATTATTA motif and interacts with the ABI
leucine zipper (bZIP) transcription factor ABRE- protein phosphatase, which suggests its role in ABA
binding proteins (AREB)/ABRE-binding factors response and stomata closure. HB6 is thought to
(ABF) bind to ABREs and activate ABA-dependent function in the ABA response in drought stress.
gene expression.36) Activation of the AREB1/ABF2 Recently, we reported other transcription factors
(AREB1) and AREB2/ABF4 (AREB2) proteins involved in abiotic stress responses. For example, a
requires ABA-mediated posttranslational phospho- drought-inducible TCP13 gene is involved in mor-
rylation. ABA-activated protein kinases, including phological changes in leaves during drought stress.47)
SNF1-related protein kinase 2 (SnRK2), phosphor- The TCP13 knockout mutant showed ABA-insensi-
ylate and activate AREB/ABF-type proteins.37),38) tive root growth and reduced dehydration-inducible
The perception of ABA and activation of AREB/ gene expression. TCP13 is thought to function in
ABFs is discussed in a later section on ABA both auxin and ABA responses in drought stress.
signaling. Three bZIP transcription factors, bZIP60, bZIP17,
and bZIP28, function in the unfolded protein
Other transcription factors involved in drought
response or ER stress responses to abiotic stress.
stress-responsive gene expression
Overexpression of bZIP60 improved salinity stress
The induction of the drought-inducible Arabi- resistance.48) Double knockout mutants of bZIP17
dopsis gene RD22, which encodes a protein with and bZIP28 showed a short-root phenotype.49)
homology to an unidentified seed protein, is mediated Suppressor mutation in TAF12b recovered root
by ABA. A MYC transcription factor, AtMYC2, and growth of the double knockout mutant.50)
a MYB transcription factor, AtMYB2, bind the Transcription factors that are involved in abiotic
RD22 promoter and cooperatively activate RD22.39) stress responses are summarized in Table 1.
These transcription factors are synthesized after
Roles of DREB2 in the crosstalk between
endogenous levels of ABA accumulate, which in-
drought and heat stress
dicates their role in a late stage of the drought
response. Overexpression of both AtMYC2 and In the heat stress responses of various organisms,
AtMYB2 not only caused an ABA-hypersensitive heat shock proteins (HSPs) function as molecular
phenotype but also improved osmotic stress toler- chaperons to protect cells from heat stress. Heat
ance.40) AtMYC2 is also a major transcription factor shock transcription factors (HSFs) function in the
in JA-induced transcription, which suggests crosstalk heat stress-responsive induction of HSPs. In plants,
between ABA and JA responses via the AtMYC2 HSFA1s function as master regulators to regulate
transcription factor.41) downstream transcription factors, including
The EARLY RESPONSIVE TO DEHYDRA- DREB2A and other HSFs (Fig. 3).13) We found
TION 1 (ERD1) gene encoding a Clp protease that DREB2A is rapidly induced by heat stress in
No. 8] Regulatory gene networks in plant abiotic stress responses 477

addition to drought stress responses.51) Domain MYB-like transcription factors regulate the expres-
analysis of DREB2A revealed that a negative sion of DREB1A and DREB1C in response to both
regulatory domain (NRD) located in the central rapid and gradual temperature decreases.14),56),57)
region of DREB2A is important for the regulation of The MYC-like transcription factor ICE1 was
DREB2A transcriptional activity.52) Transgenic reported to induce the DREB1A/CBF3 promoter
plants overexpressing constitutively active forms of based on the analysis of an ice1-1 mutant. However,
DREB2A (DREB2A CA) that lack the NRD show DREB1A/CBF3 repression in the ice1-1 mutant
the enhancement of both stress-inducible gene was not correlated with ICE1 mutation. ICE1 is
expression and dehydration stress resistance. The identical to SCRM1 in stomatal development but is
stability of the DREB2A protein is regulated by not involved in the cold stress response. Therefore,
modification of the NRD. DREB2A-interacting many reports on the ICE1-DREB1A/CBF3 regula-
protein 1 (DRIP1) and DRIP2 were isolated as E3 tory model in the cold stress response need to be
ubiquitin ligases that mediate the ubiquitination of revalidated without preconceptions.58)–60) More de-
DREB2A.53) Ubiquitinated DREB2A is degraded tailed analysis of the regulatory systems of cold-
through the 26S proteasome pathway in control responsive transcription are reviewed in Kidokoro
conditions. et al. (2022).14)
The DREB2A protein is regulated by other
Metabolomics analysis of plant abiotic
degradation pathways in heat stress conditions.
stress responses
BTB/POZ and MATH domain proteins (BPMs)
encode substrate adaptors of the CULLIN3-based E3 Integrated analyses of transcriptome and me-
ligase and bind to the NRD of DREB2A.54) The tabolomics analyses in Arabidopsis revealed connec-
inhibition of phosphorylation in NRD by CASEIN tions between genes and primary metabolites in
KINASE 1 (CK1) stabilizes and activates DREB2A dehydration conditions. Metabolite profiling by gas-
in response to heat stress.55) DREB2A mediates chromatography-mass spectrometry and capillary
drought and heat stress signaling with the different electrophoresis-mass spectrometry analyses revealed
regulatory mechanisms of NRD in DREB2A. The that ABA accumulates during dehydration, regulat-
heat stress responses were reviewed in detail by ing the accumulation of various amino acids and
Ohama et al. (2017).13) sugars such as glucose and fructose.61) In particular,
the dehydration-inducible accumulation of branch-
Regulation of DREB1s/CBFs induction
chain amino acids, saccharopine, proline, and agma-
in response to cold stress
tine is correlated with the dehydration-inducible
DREB1s/CBFs bind to the DRE cis-acting expression of their key biosynthetic genes, such as
element and act as master regulators in several branch-chain aminotransferase 2 (AtBCAT2), lysine
cold-inducible transcription pathways. There are ketoglutarate reductase/saccharopine dehydrogenase
three DREB1/CBF genes in Arabidopsis: (AtLKR/SDH), Pyrroline-5-carboxylate synthase 1
DREB1B/CBF1, DREB1A/CBF3, and DREB1C/ (AtP5CS1), and arginine decarboxylase 2 (AtADC2),
CBF2, which are arrayed in this order. They are all respectively. These genes are induced by endogenous
rapidly induced by cold stress. Arabidopsis mutant ABA in drought stress conditions.
plants in which three DREB1 genes are disrupted Metabolites that are involved in abiotic stress
show decreased freezing tolerance (Fig. 3). response and tolerance are summarized in Table 2.
Two different types of transcription factors are In contrast, the biosynthesis pathway of raffi-
involved in cold-inducible DREB1 expression, name- nose family oligosaccharides (RFOs) is controlled by
ly, the Calmodulin-Binding Transcriptional Activa- the ABA-independent pathway. Galactinol synthase
tor (CAMTA) and circadian clock-related MYB-like (GolS) is a key enzyme involved in galactinol and
transcription factors such as CIRCADIAN CLOCK raffinose biosynthesis.62) RFO protects cells from
ASSOCIATED 1 (CCA1), LATE ELONGATED oxidative stress.63) The AtGolS1 and AtGolS2 genes
HYPOCOTYL (LHY) and REVEILLEs (RVEs). encoding galactinol synthase are induced by dehy-
Plants recognize cold stress as two different types dration in an ABA-independent pathway. Over-
of signals, namely, rapid or gradual temperature expression of AtGolS2 increased drought stress
decreases.56),57) CAMTAs respond to a rapid tem- tolerance, which has been applied to the development
perature drop, strongly inducing the expression of of drought-tolerant crops in the field.64) The .-amino-
DREB1B and DREB1C. In contrast, clock-related butylate (GABA) shunt pathway to produce succi-
478 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

Table 2. Names of hormones and metabolites involved in abiotic stress responses and tolerance

Metabolite Functions Abiotic stress References

Plant hormones involved in stress responses
ABA Abscisic acid Dry 5, 66, 104
ABA-Glu Abscisic acid glucosyl ester Dry 66, 104
JA Jasmonic acid Dry, Wound 66
JA-Ile Jasmonic acid isoleucine Dry 66
SA Salicylic acid Dry, Wound 66
Cytokinin Kinetin, Zeatin Dry 66

Metabolites involved in abiotic stress tolerance

Pro Proline Dry, ABA 7, 61
RFO Raffinose family oligosaccharides Dry, Cold, Heat 7, 61, 62
(galactinol, raffinose)
BCAA Branch-chain amino acid Dry, ABA 7, 61
(valine, leucine, isoleucine)
Saccharopine Dry, ABA 7, 61
Agmatine Dry, ABA 7, 61
GABA .-amino-butyric acid ABA (!) 7, 61
Alanine ABA (!) 7, 61
Succinate ABA (!) 7, 61
Plant hormones and metabolites involved in abiotic stress responses and tolerance are listed in this table and discussed in the text.
Dry: drought stress; Cold: cold stress; Heat: heat stress; ABA: abscisic acid regulated; Wound: wound stress. (!): negative regulation.

nate from glutamate is negatively controlled by the drought stress response, there seems to be
ABA. GABA, alanine, and succinate are negatively crosstalk between ABA and JA-Ile.
regulated by ABA. GABA decarboxylase GAD4 is Plant hormones involved in abiotic stress
involved in this process and ABA repression.61) responses are summarized in Table 2.
Metabolomics analysis of transgenic Arabidopsis
ABA perception and signaling in the
overexpressing AtDREB1A/CBF3 revealed an ABA-
abiotic stress response
independent metabolic network with similarity to
the cold-induced metabolomics profile.65) Overex- The perception and signal transduction in ABA
pression of AtDREB1A/CBF3 significantly increased responses have been thoroughly studied.3),5) ABA-
cold stress tolerance. However, overexpression of insensitive mutants abi1 and abi2 were isolated, and
AtDREB2A in transgenic plants increased their both ABI1 and ABI2 genes encode Protein Phospha-
tolerance to dehydration stress but only slightly tase 2C (PP2C), which suggests an important role
increased freezing stress tolerance.51) The increased of protein phosphorylation in ABA signaling. Then,
tolerance to freezing stress in transgenic AtDREB1A- the protein kinase SnRK2 was shown to be a major
overexpressing plants depends on the accumulation protein kinase family in ABA signaling.67)–70) SnRK2
of low-temperature-regulated metabolites, especially family genes have 10 members and are classified into
raffinose and galactinol. 3 subclasses. Among them, 3 genes of subclass III
Profiles of plant hormones were analyzed using SnRK2 play important roles in ABA signal trans-
dehydrated Arabidopsis plants in moderate dehydra- duction because their triple knockout mutant showed
tion stress.66) ABA is a major plant hormone in the severe ABA-insensitive phenotypes.71),72) Subclass III
drought stress response. Jasmonic acid isoleucine SnRK2s interact with PP2Cs and phosphorylate
(JA-Ile) increased slightly in wild-type plants and AREB/ABF transcription factors to induce ABA-
significantly increased in nced3 mutants in response inducible genes.37),69) These observations support the
to dehydration. Salicylic acid and cytokines are important roles of SnRK2 and PP2C in ABA signal
decreased during the late dehydration response. In transduction pathways.
No. 8] Regulatory gene networks in plant abiotic stress responses 479

To understand ABA action, the identification of Phosphoproteomics analysis using triple mu-
receptors is necessary. After long-term efforts by tants of subclass III SnRK2s revealed their down-
many groups, PYR/PYL/RCAR-type or START- stream phosphorylated target proteins, which are
type soluble ABA receptors were identified using bZIP transcription factors, including AREB1/ABF2,
chemical genetic and biochemical approaches.73),74) AREB3, and EEL.79) mitogen-activated protein
Biochemical and genetic analyses revealed that these kinases (MAPKs) MPK1 and MPK2 are targets of
proteins directly bind PP2Cs in the presence of ABA SnRK2. Unknown proteins, including SNS1, were
and inhibit their activity in an ABA-dependent identified as SnRK2 target proteins.
manner. This finding indicates that ABA regulates Subclass III SnRK2s are activated by MAPK
cellular processes by modulating PP2C activity. kinase (MAPKKK) or B-type Raf-like kinase in
Biochemical analysis showed that clade-A PP2Cs Physcomitrellla patens.80) In Arabidopsis, B-type
interact with ABA-activated SnRK2s and inactivate Raf-like protein kinases also function in the phospho-
them efficiently through dephosphorylation at spe- rylation and activation of subclass III SnRK2. B2-
cific amino acid residues.75) Because SnRK2s can and B3-Raf kinases are activated by osmotic stress in
phosphorylate and activate some ABA-dependent an ABA-independent manner, which indicates cross-
transcription factors (see above), one of the ABA talk between ABA-dependent and ABA-independent
signaling pathways is now established from percep- activation of SnRK2 (Fig. 4).81)–83) In contrast,
tion to gene expression, namely, PYR/PYL/RCAR- subclass I SnRK2s are activated by dehydration
related receptor ! clade-A PP2C ! ABA-activated stress in an ABA-independent pathway. Dehydra-
SnRK2 ! AREB/ABF bZIP transcription factors tion-activated subclass I SnRK2s regulate mRNA
(Fig. 4).75)–78) decay by phosphorylating VARICOSE (VCS), which

Drought stress
ABA-independent ABA
pathway
PYR/PYL/RCAR
B4-Raf-like kinase ABA
(MAPKKK) PP2C B2-, B3-Raf-like
kinase
(MAPKKK)
Subclass I Subclass III & II
SnRK2 SnRK2
Phosphorylation

VARICOS(VCS) AREB/ABF
mRNA decapping enzyme SLAC
TF
Degradation Transcription Stomata
of mRNA of stress genes closure
Recycling of unnecessary mRNAs Proteins involved in stress tolerance
Fig. 4. Cellular phosphorylation signaling in ABA-dependent and ABA-independent responses in drought stress. SnRK2 protein kinases
are major protein kinases involved in drought stress responses. Subclass III SnRK2s function in the ABA-dependent pathway
downstream of PYR/PYL/RCAR ABA receptors and the protein phosphatase PP2C. Upstream of SnRK2, Raf-like kinases function
as activators of SnRK2 in drought stress conditions. Activated SnRK2s phosphorylate and activate AREB/ABF transcription factors
to upregulate stress-inducible genes. In contrast, subclass I SnRK2 functions in the ABA-independent pathway to regulate
downstream VCS, which regulates mRNA degradation. This process functions in the recycling of ribonucleotides for the induction of
stress genes. Names and functions of genes for transcription factors (TFs) and signaling factors (SFs) are summarized in Table 1.
480 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

is a scaffold protein of mRNA-decapping com- ent types of sensing molecules have been identified as
plexes.84) B4-Raf kinase phosphorylates subclass I possible candidates for sensors of water deficit status,
SnRK2s. Therefore, the B4-Raf kinase – subclass I namely, osmosensors, mechanosensors, calcium chan-
SnRK2 – VCS signaling system regulates the mRNA nels, and ROS sensors (Fig. 5). Combinations of
population to adapt to dehydration status. Recycling these sensors recognize water-deficit conditions to
of unnecessary mRNAs in stress conditions is properly respond to drought stress. Molecular pat-
necessary for dehydration stress responses to synthe- terns of water-deficit stress are sensed by these
size mRNAs for stress proteins. Details of the different types of sensors, and the sensed patterns
phospho-signaling pathways in the drought stress of water deficiency signals are then transmitted to
response are provided by Soma et al. (2021).11) cellular signaling networks to induce stress-specific
Signaling factors and receptors in ABA response gene expression profiles to achieve appropriate
are summarized in Table 1. responses to dehydration status. There are different
types of signaling pathways that properly control
Perception of dehydration stress signals
stress responses (Fig. 6).17)
Land plants sense water deficit status mainly in Histidine kinases have been analyzed as osmo-
roots and monitor the reduction of water potential sensors in yeast and bacteria. In yeast, Sln1p encoding
in the root vasculature. Then, the hydraulic stress a histidine kinase functions as an osmosensor up-
signal in root cells is transmitted to leaves to mediate stream of the HOG1 MAPK pathway.9) In Arabi-
stomatal closure and stress-inducible gene expression dopsis, histidine kinase 1 (ATHK1/AHK1) was
to protect plants from dehydration status.15),85),86) shown to be a functional homolog of yeast Sln1p
The sensing mechanism of hydraulic stress in roots is encoding an osmosensor histidine kinase.87) AHK1
complex and has not been clearly elucidated. Differ- was shown to function as a positive regulator in

Fig. 5. Sensing system and cellular signal transduction in drought stress responses. Drought is a complex stress for plants and induces
different types of stress responses, such as osmotic, mechanical, and oxidative stresses. These complex stress signals are sensed by
different cellular sensors and transduced to downstream signaling networks, including phosphorylation, calcium, and ROS signals.
Molecular patterns of stress signals activate certain genes, hormones, and transporters to induce proper stress responses to adapt to
drought stress conditions. Names and functions of sensors, receptors and signaling factors are summarized in Table 1.
No. 8] Regulatory gene networks in plant abiotic stress responses 481

Fig. 6. Interorgan signaling from roots to leaves in the drought stress response. Interorgan signals of water deficiency are transmitted via
the vasculature from roots to leaves. Change in turgor pressure, production of ROS, and transient calcium signals function in the early
drought stress response. Peptides and metabolites are produced in roots and transmitted to leaves via xylem. In leaves, ABA is mainly
produced in the vasculature and guard cells in response to stress signals from roots and leaves. The CLE25 peptide is synthesized in
roots in drought stress conditions, transmitted to leaves via xylem, and then induces ABA biosynthesis in leaves. Signal transport
from leaves to roots has not been analyzed yet (shown as a dashed arrow). These were also discussed before.17) This figure is reprinted
with modification from Ref. 17.

drought stress tolerance by the analysis of its mu- The calcium current across the plasma membrane is
tant and overexpressors.88),89) Other AHKs, AHK2, activated by various stress signals, including osmotic
AHK3, and AHK4 (CRE1), function as negative stress (Fig. 5). Ca2D waves rapidly transmit stress
regulators through cytokinin regulation.90) There may information to distant tissues. REDUCED
be complex osmosensing systems, including AHK1 HYPEROSMOLALITY INDUCED Ca2D IN-
and downstream signaling MAPKs, in plants because CREASE 1 (OSCA1) functions as a channel protein
plant MAPKs are activated not only by abiotic stress that mediates Ca2D influx in the osmotic stress
signals but also by biotic stress signals to control response.96)–98) The role of OSCA1 in stomatal
complex downstream events, including gene expres- closure is mediated only by osmotic stress-mediated
sion and physiological responses (Fig. 5).91) Ca2D influx in stomata. CALCIUM-PERMEABLE
Stretch-activated calcium channels are thought STRESS-GATED CATION CHANNEL 1 (CSC1)/
to function as osmosensor candidates in drought OSCA1.2 functions as a hyperosmolality-gated cal-
stress sensing. In plants, calcium-permeable mecha- cium-permeable channel protein and mediates Ca2D
nosensitive channels (MCAs) 1 and 2 sense osmotic influx.99),100) Therefore, OSCA family membrane
changes in the plasma membrane to mediate Ca2D proteins can mediate Ca2D influx in dehydration
influx to activate downstream cellular signaling.92),93) stress responses.
MCA1 is a homolog of yeast MID1 and functions as HYDROGEN-PEROXIDE-INDUCED Ca2D
a Ca2D-permeable stretch-activated channel compo- INCREASE (HPCA) was reported as a sensor of
nent in plants.94),95) MCA1 and 2 are other candidate H2O2 in guard cells.101) HPCA1, a leucine-rich repeat
osmosensors for complex drought stress responses. receptor-like kinase, is activated by H2O2 in the
482 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

extracellular domain, and induces Ca2D influx in intercellular transport of signaling molecules through
guard cells for stomatal closure (Fig. 5). ROS cellular membranes. Different types of mem-
signaling functions in response to various types of brane transporters function as ABA transporters
environmental stresses, including dehydration. ROS (Fig. 6).110) Among them, ATP binding cassette
signaling is thought to function in complex drought (ABC) transporter genes were shown to function as
conditions. The ROS wave is induced through ABA transporters. There are 129 ABC transporter
respiratory burst oxidase D (RBOHD) and regulates genes in Arabidopsis. Among them, a AtABCG25 Ds-
stomatal closure of stressed leaves. ROS signaling tagged knockout mutant showed an ABA-sensitive
has important roles in coordinating systemic re- phenotype.111) AtABCG25 encodes a half-size ABC
sponses in different leaves to extreme abiotic stress- transporter that belongs to the WBC subfamily. The
es.102),103) ROS are also involved in ABA signaling. AtABCG25 protein was localized on the plasma
Sensors and receptors that are involved in membrane. ABCG25 is significantly expressed in
abiotic stress perception are summarized in Table 1. phloem companion cells. Biochemical analysis
showed that AtABCG25 functions as an ABA
Biosynthesis and transport of ABA
exporter. Water loss of the ABCG25-overexpressing
in intertissue signaling
plants was much less than that of wild-type
In salinity or drought stress conditions, accu- plants.112) AtABCG25-overexpressing plants showed
mulated ABA has pivotal roles in stress responses higher water-use efficiency than wild-type plants.113)
in plants through a drastic change in the gene Another ABC transporter, AtABCG40, was shown
expression profile and cellular processes. Our knowl- to function as an ABA importer.114) AtABCG40 is
edge of the synthesis and metabolism of ABA has expressed in guard cells. This is consistent with the
greatly increased.6),104) In particular, the identifica- hypothesis that ABA is a mobile signal between
tion of rate-limiting enzymes such as 9-cis-epoxycar- vascular tissues and epidermal tissues, including
otenoid dioxygenases (NCEDs) in the ABA biosyn- guard cells, that controls stomatal regulation. In
thetic pathway and P450 CYP707s in the ABA addition, AtABCG22 was shown to function in
catabolic pathway has greatly enhanced our under- stomatal closure.115) Its knockout mutant shows open
standing of how plants control the level of this stomata and a dehydration-sensitive phenotype.
phytohormone. The genes for these two enzymes are AtABC22 is expressed in guard cells. However, the
activated by various stress treatments; NCED3 is molecule transported by AtABCG22 has not yet
strongly induced by dehydration and downregulated been identified.
by rehydration, whereas CYP707A is further upre- In addition to the ABC transporter family, ABA
gulated during rehydration, suggesting fine-tuning transporters in other families of membrane trans-
of the ABA level by the coordinated regulation of porters have been reported (Fig. 6). Arabidopsis
ABA production and breakdown systems.105),106) In NPF4.6 and several members of the NITRATE
addition, ABA is stored as an inactivated form, ABA TRANSPORTER 1/PEPTIDE TRANSPORTER
glucosyl ester, in vacuoles and apoplasts. In dehy- FAMILY (NPF) have been identified as ABA trans-
dration conditions, ABA is released from the glucosyl porters.116)–118) NPF4.6 and NPF5.1 act as ABA
ester form by O-glucosidase.107) Genes involved in importers. NPF4.6 functions in guard cells as well as
ABA biosynthesis are strongly expressed in vascular vascular tissues, and NPF5.1 is expressed in leaf
tissues. Among them, the drought-inducible NCED3 cells. These NPFs are involved in stomatal closure by
gene is expressed strongly in vascular tissues and regulating the amount of ABA. Arabidopsis thaliana
induces ABA accumulation in leaves.108) NCED3 is DETOXIFICATION EFFLUX CARRIER 50
regulated by an NGA1 transcription factor with a (AtDTX50), which belongs to the multidrug and
B3 domain.109) Other genes involved in ABA biosyn- toxin efflux transporter family, has been identified
thesis are also expressed in vascular tissues, which as another ABA transporter.119) The AtDTX50
means ABA synthesis mainly occurs in vascular protein has ABA exporter activity and localizes to
tissues.6),17) In guard cells, genes involved in ABA the plasma membrane. AtDTX50 is preferentially
biosynthesis are also expressed and function in expressed in both vascular tissues and guard cells.
stomatal closure in rapid response to water-deficit Atdtx50 mutant plants are more tolerant to drought
conditions (Fig. 6). with lower stomatal conductance. AtDTX50 can also
ABA is a mobile signal molecule in plant stress modify ABA transport in both vascular and guard
responses. Membrane transporters function in the cells. Several ABA transport systems have been
No. 8] Regulatory gene networks in plant abiotic stress responses 483

reported to date. Details of the different functions of functions in the adaptation to water deficiency to
these ABA transporter systems and their coordinated control plant growth in drought stress conditions.
roles are discussed by Kuromori et al. (2018).110) Precise reviews on mobile peptides were reported
ABA related transporters are summarized in by Takahashi et al. (2020)7) and Yoshida et al.
Table 1. (2021).16)
CLE25 peptides mediating drought Phenotyping of stress tolerance
stress responses in local and
To understand the complex mechanisms under-
long-distance signaling
lying plant responses to environmental stresses at
Precise analyses of the Arabidopsis genome have the whole plant level, it is important to measure
revealed the existence of many small open reading plant growth in environmental stress conditions by
frames encoding peptides. These predicted small open controlling growth conditions (Fig. 7). A quantita-
reading frames are transcribed in plants, which tive phenotyping system facilitates automated meas-
suggests the existence of small peptides that function urements of plant growth by regulating the timing
as hormones in various stages of plant development. and strength of adverse environmental stimuli and
Plant peptides mediate regulatory roles in cell-to-cell conducting time-course observations of multiple pa-
communication and long-distance signaling, such as rameters of plant growth in stress conditions. Several
phytohormones.7),16),120) As a functional feature of phenotyping systems have been established recently
those mobile molecules, peptides are perceived by at different organizational scales. Automated pheno-
each peptide-specific receptor in each tissue, enabling typing systems provide powerful tools for plant
peptide–receptor modules to precisely transmit research by employing large-scale, high-throughput,
environmental stimuli from sensing tissues to target and nondestructive systems to analyze time-depend-
tissues. The family of CLAVATA3/EMBRYO- ent changes in plant growth (Fig. 7).122)–124) We
SURROUNDING REGION-RELATED (CLE) pep- combined the advantages of existing automated
tides include well-characterized peptides active in platforms to construct the RIKEN Integrated Plant
developmental processes. We showed that one of the Phenotyping System (RIPPS) with automated
CLE peptides, CLE25, functions as a mobile root-to- weighing and watering stations and continuous
shoot signaling molecule that modulates stomatal rotation of plants during the experiment to unify
closure and the expression of stress-related genes in growth conditions.125) Two major benefits of this
leaves in dehydrated soil (Fig. 6).121) CLE25 gene system are (1) the microenvironmental conditions
expression is enhanced in root vascular tissues in experienced by individual plants are homogenized
response to dehydration stress. Root vasculature- and (2) additional imaging devices can be easily set
derived CLE25 peptide moves from the roots to the to any position. With the RIPPS, we can obtain
leaves and induces NCED3 expression to enhance digital images of individual plants in the daytime
ABA accumulation in the leaves. In leaves, BAM1 and during the nighttime dark conditions using
and BAM3 receptor-like kinases function as receptors infrared (IR) LED light (peak wavelength 950 nm).
of the CLE25 peptide and transmit the signal to We used an IR camera to measure leaf temperature
induce NCED3 to accumulate ABA, which then and a near IR camera to measure the water status of
regulates the stomatal aperture (Fig. 6). Taken leaves. By using these different cameras, plant status
together, plants sense water deficit conditions in can be measured more precisely in environmental
their roots and integrate the information among stress conditions. Time-dependent changes in plant
distant organs to optimize stress adaptations and size and detailed analyses of temporal and spatial
resistance in whole plant tissues. effects can be derived and quantified from time-lapse
Systematic analyses revealed that almost all growth images. The RIPPS phenotyping platform is
CLE peptide genes are expressed in roots. Exogenous operated with the precise control of growth parame-
applications of synthetic CLE peptides inhibit root ters, including temperature, light, nutrient availabil-
growth elongation, indicating that several CLE ity, and water conditions. We used the RIPPS to
peptides, including CLE25, redundantly mediate establish quantitative evaluation methods for deter-
the regulation of root development. Cle25 mutants mining the growth responses and water use efficien-
show a normal root growth phenotype. The root- cies of Arabidopsis plants in different water con-
expressed CLE25 transmits information on water- ditions.126) We also analyzed the involvement of
deficit stress from the roots to leaves, and CLE25 ABA metabolism in water use efficiency using the
484 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

Fig. 7. Translation of basic knowledge on plant responses to drought stress for the application on molecular breeding of crops with higher
yields in drought stress conditions. In cellular stress responses, many useful genes have been identified, such as transcription factors,
protein kinases, ABA-related regulators, and many stress-inducible genes. Moreover, interorgan signaling molecules have also been
discovered. By using these useful genes, genome editing technology and genetic modified organism technology can be used for the
development of drought-tolerant crops. Phenotyping and data science will provide useful technology for systematic breeding and
cultivation technologies for crops and vegetables in the future. The RIKEN Integrated Plant Phenotyping System (RIPPS) was
reported by Fujita et al.125) and its operation can be seen in the website of RIKEN CSRS in the following URL. http://
www.csrs.riken.jp/jp/virtualtour/yokohama/index.html?onstartFloadscene(scene9,null.MERGE,BLEND(1.2)).

RIPPS.125) These proof-of-concept studies demon- growth, and food production will need to increase
strate that the RIPPS accurately facilitates the by more than 1.6-fold by 2050. Increasing the
measurement and evaluation of plant growth re- production of agricultural products has become an
sponses to precisely controlled hydric environments. urgent issue. However, many fields have become dry
By using a quantitative phenotyping system, we can and poorly nourished because of global warming.
analyze gene knockout mutants and natural genetic Therefore, there is a demand for the development of
variants to perform high-throughput genetic map- crops that are resistant to drought and have higher
ping and large-scale analysis of gene expression yields with less water (Fig. 7).2),12)
pathways, metabolite levels, or plant hormone levels. With such expectations, we have been con-
Gene discovery of novel factors involved in abiotic ducting joint research with the Japan Interna-
stress responses can be achieved by improving tional Research Center for Agricultural Research
phenotyping accuracy and throughput. A precise (JIRCAS), the International Center for Tropical
review on phenotyping of drought tolerance was Agriculture (CIAT) in Colombia, the International
provided by Kuromori et al. (2022).17) Rice Research Institute (IRRI) in the Philippines,
and the International Maize and Wheat Improve-
Molecular breeding of environmental
ment Center (CIMMYT) in Mexico.127),128) This
stress tolerance
international collaboration was supported by a
The world’s food supply will need to increase due special fund (2007–2012) from the Ministry of
to the world’s population growth and economic Agriculture, Forestry and Fisheries (MAFF), Japan.
No. 8] Regulatory gene networks in plant abiotic stress responses 485

We tested Arabidopsis and rice stress-related genes more than 30 years of research and development.
and various promoters. The stress genes used were Some of the genes identified have been applied to
transcription factors such as DREBs and AREBs, improve the drought tolerance of crops and their
NCED3 in ABA synthesis, LEA proteins and productivity in drought conditions.
metabolic enzymes involved in stress resistance. We have discovered sophisticated regulatory
The promoters used are constitutive promoters and systems for plant responses and tolerance to drought
drought-inducible promoters. Among them, over- and temperature stresses and their crosstalk in
expression of the AtGolS2 gene in combination with regulatory networks. It is still unclear how plants
the ubiquitin promoter rendered transgenic rice recognize complex environmental changes and stress
more resistant to drought and increased grain yield to respond and adapt to survive in severe stressful
in drought conditions in the field. AtGolS2 is an conditions. We realized that plants have various
Arabidopsis gene encoding galacitinol synthase, a key types of sensors and signaling systems to recognize
enzyme in RFO biosynthesis. RFO has a function in the molecular patterns of these stress signals in
removing active oxygen.62),63) AtGolS2-overexpress- response to environmental changes and stresses.
ing rice plants had higher relative water content in Molecular patterns of stress signals are transferred
the leaves and drought tolerance. Cultivation of to properly induce downstream molecular events. In
transgenic rice in a dry field in Columbia demon- the coming decades, it will be important to under-
strated that introduction of the AtGolS3 gene is stand the molecular patterns of stress recognition,
effective in developing drought-resistant and high- signaling networks, and stress responses in complex
yielding crops in dry fields.64),128) We also reported environmental conditions (Fig. 7).17)
that other genes, such as transcription factors To understand plant responses to environmental
DREBs and AREBs, are also useful for the breeding stress, it is important to monitor plant phenotyping
of drought tolerance and higher yield in dry fields.128) quantitatively by using imaging technology in combi-
We collaborated with Embrapa (Brazilian Cor- nation with information technology. These pheno-
poration of Agricultural Research) to test Arabidop- type data in various environmental conditions will be
sis stress genes in transgenic soybeans in greenhouses useful to evaluate plant performance in various stress
and dry fields. This project was supported by the conditions. Moreover, monitoring plant growth in
Science and Technology Research Partnership for controlled environmental conditions can provide
Sustainable Development (SATREPS) of the Japan useful tools for quantitative phenotyping. In combi-
Science and Technology Agency (JST) and Japan nation with omics analyses such as transcriptomics
International Cooperation Agency (JICA) from 2009 and metabolomics analyses, plant status in certain
to 2014.128) Transgenic soybean expressing Arabidop- conditions can be precisely described (Fig. 7). We
sis GolS2, DREB, AREB, NCED3, and other genes can access various mutant resources, including gene
showed improved drought tolerance in the green- knockout lines and ecotypes in Arabidopsis, which
house. We also tested transgenic soybean lines in dry provide useful and precise data on plant responses to
fields in Brazil.129) Some of these useful genes have abiotic stress. By applying these methods to various
also been applied to sugarcane, which has yielded plants and crops, we can analyze their responses at
good results and been applied to field trials.130) the molecular level. By integrating genomics and data
science, we can describe profiles of plant responses
Summary and perspectives
and crop performance in various combinations of
Since 1989, we have been studying complex environmental stresses in the future.131)
plant responses to abiotic stress conditions such as
Acknowledgments
drought, heat, and cold stresses. We used functional
genomics approaches to discover important genes We would like to thank all the members of our
and factors involved in gene expression and signal laboratories and collaborators for their valuable
transduction in response to drought, heat, and cold contributions. We also thank RIKEN, The Univer-
stress conditions. We have identified key transcrip- sity of Tokyo, and Japan International Research
tion factors in stress-inducible gene expression; Center for Agricultural Sciences (JIRCAS) for their
important phosphorylation processes in stress signal- support of our research. We also thank the Japan
ing; key genes in ABA synthesis, degradation, and Society for the Promotion of Science (JSPS), Bio-
transporter; peptides involved in long-distance sig- oriented technology Research Advancement Institu-
naling; and metabolites for stress tolerance during tion (BRAIN), and Japan Science and Technology
486 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

Agency (JST) for funding our research. We apologize 14) Kidokoro, S., Shinozaki, K. and Yamaguchi-
for not referencing all manuscripts on related topics Shinozaki, K. (2022) Transcriptional regulatory
network of plant cold-stress responses. Trends
due to space limitations. Plant Sci. S1360–S1385.
15) Takahashi, F., Hanada, K., Kondo, T. and
References Shinozaki, K. (2019) Hormone-like peptides and
small coding genes in plant stress signaling and
1) Shinozaki, K., Uemura, M., Bailey-Serres, J., Bray, development. Curr. Opin. Plant Biol. 51, 88–95.
A.E. and Weretilnyk, E. (2015) Responses to 16) Yoshida, T., Fernie, A.R., Shinozaki, K. and
abiotic stress. In Biochemistry and Molecular Takahashi, F. (2021) Long-distance stress and
Biology of Plants. 2nd ed. (eds. Buchanan, B., developmental signals associated with abscisic
Gruissem, W. and Jones, R.L.). Wiley & Black- acid signaling in environmental responses. Plant
well, Chichester, pp. 1051–1100. J. 105, 477–488.
2) Gupta, A., Rico-Medina, A. and Cafio-Delgado, A.I. 17) Kuromori, T., Fujita, M., Takahashi, F.,
(2020) The Physiology of plant responses to Yamaguchi-Shinozaki, K. and Shinozaki, K.
drought. Science 368, 266–269. (2022) Inter-tissue and Inter-organ signaling in
3) Cutler, S., Rodriquez, P.L., Finkelstein, R.R. and drought stress responses and phenotyping of
Abrams, S.R. (2010) Abscisic acid: emergence of a drought tolerance. Plant J. 109, 342–358.
core signaling network. Annu. Rev. Plant Biol. 18) Yamaguchi-Shinozaki, K., Koizumi, M., Urao, S.
61, 651–659. and Shinozaki, K. (1992) Molecular cloning and
4) Hirayama, T. and Shinozaki, K. (2010) Research on characterization of 9 cDNAs for genes that are
plant abiotic stress responses in the post-genome responsive desiccation in Arabidopsis thaliana:
era: past, present and future. Plant J. 61, 1041– Sequence analysis of one cDNA clone that encodes
1052. a putative transmembrane channel protein. Plant
5) Yoshida, T., Christman, A., Yamaguchi-Shinozaki, Cell Physiol. 33, 217–224.
K., Grill, E. and Fernie, A.R. (2019) Revisiting 19) Kiyosue, T., Yamaguchi-Shinozaki, K. and
the basal role of ABA-Roles outside of stress. Shinozaki, K. (1994) Cloning cDNAs for genes
Trends Plant Sci. 24, 625–635. that are early-responsive to dehydration stress
6) Chen, K., Li, G.-J., Bresson, R.A., Song, C.-P., Zhu, (ERDs) in Arabidopsis thaliana L.: identification
J.-K. and Zao, Y. (2020) Abscisic acid dynamics, of three ERDs as HSP cognate genes. Plant Mol.
signaling, and functions in plants. J. Intgr. Plant Biol. 25, 791–798.
Biol. 62, 25–54. 20) Seki, M., Narusaka, M., Abe, H., Kasuga, M.,
7) Takahashi, F., Kuromori, T., Urano, K., Yamaguchi-Shinozaki, K., Carninci, P. et al.
Yamaguchi-Shinozaki, K. and Shinozaki, K. (2001) Monitoring the expression pattern of 1300
(2020) Drought-stress responses and resistance in Arabidopsis genes under drought and cold stresses
plants: From cellular responses to long-distance by using a full-length cDNA microarray. Plant
intercellular communication. Front. Plant Sci. 11, Cell 13, 61–72.
556972. 21) Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita,
8) Zhang, H., Zhu, J., Gong, Z. and Zhu, J.-K. (2022) M., Oono, Y. et al. (2002) Monitoring the
Abiotic stress responses in plants. Nat. Genet. 23, expression profiles of 7000 Arabidopsis genes
104–119. under drought, cold and high-salinity stresses
9) Shinozaki, K. and Yamaguchi-Shinozaki, K. (1997) using a full-length cDNA microarray. Plant J. 31,
Gene expression and signal transduction in water- 279–292.
stress response. Plant Physiol. 115, 327–334. 22) Yamaguchi-Shinozaki, K. and Shinozaki, K. (1993)
10) Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Characterization of a desiccation-responsive rd29
Transcriptional regulatory networks in cellular gene of Arabidopsis thaliana and analysis of its
responses and tolerance to dehydration and cold promoter in transgenic plants. Mol. Gen. Genet.
stresses. Annu. Rev. Plant Biol. 57, 781–803. 236, 331–340.
11) Soma, F., Takahashi, F., Yamaguchi-Shinozaki, K. 23) Hajela, R.K., Horvath, D.P., Gilmour, S.J. and
and Shinozaki, K. (2021) Cellular phosphorylation Thomashow, M.F. (1990) Molecular cloning of cor
signaling and gene expression in drought-stress (cold-regulated) genes in Arabidopsis thaliana.
responses: ABA-dependent and ABA-independent Plant Physiol. 93, 1246–1252.
regulatory systems. Plants 10, 756. 24) Nordin, K., Vahala, T. and Palva, E.T. (1993)
12) Umezawa, T., Fujita, M., Fujita, Y., Yamaguchi- Differential expression of two related, low-temper-
Shinozaki, K. and Shinozaki, K. (2006) Engineer- ature-induced genes in Arabidopsis thaliana (L.).
ing drought tolerance in plants: discovering and Heynh. Plant Mol. Biol. 21, 641–653.
tailoring genes to unlock the future. Curr. Opin. 25) Seki, M., Umezawa, T., Urano, K. and Shinozaki, K.
Biotechnol. 17, 113–122. (2007) Regulatory metabolic networks in drought
13) Ohama, N., Sato, H., Shinozaki, K. and Yamaguchi- stress responses. Curr. Opin. Plant Biol. 10, 296–
Shinozaki, K. (2017) Transcriptional regulatory 302.
network of plant heat stress response. Trends 26) Nakaminami, K., Matsui, A., Shinozaki, K. and
Plant Sci. 22, 53–65. Seki, M. (2012) RNA regulation in plant abiotic
No. 8] Regulatory gene networks in plant abiotic stress responses 487

stress responses. Biochim. Biophys. Acta 1819, activator AREB1. Proc. Natl. Acad. Sci. U.S.A.
149–153. 103, 1988–1993.
27) Khraiwesh, B., Zhu, J.-K. and Zhu, J. (2012) Role of 38) Yoshida, T., Fujita, Y., Sayama, H., Kidokoro, S.,
miRNAs and siRNAs in biotic and abiotic stress Maruyama, K., Mizoi, J. et al. (2010) AREB1,
responses in plants. Biochim. Biophys. Acta 1819, AREB2, and ABF3 are master transcription
137–148. factors that cooperatively regulate ABRE-de-
28) Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) pendent ABA signaling involved in drought stress
A novel cis-acting element in an Arabidopsis gene tolerance and require ABA for full activation.
is involved in responsiveness to drought, low- Plant J. 61, 672–685.
temperature, or high-salt stress. Plant Cell 6, 39) Abe, H., Yamaguchi-Shinozaki, K., Urao, T.,
251–264. Iwasaki, T., Hosokawa, D. and Shinozaki, K.
29) Baker, S.S., Wilhelm, K.S. and Thomashow, M.F. (1997) Role of Arabidopsis MYC and MYB
(1994) The 5B-region of Arabidopsis thaliana homologs in drought- and abscisic acid-regulated
cor15a has cis-acting elements that confer cold-, gene expression. Plant Cell 9, 1859–1868.
drought- and ABA-regulated gene expression. 40) Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K.
Plant Mol. Biol. 24, 701–713. and Yamaguchi-Shinozaki, K. (2003) Arabidopsis
30) Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, AtMYC2 (bHLH) and AtMYB2 (MYB) function
S., Yamaguchi-Shinozaki, K. et al. (1998) Two as transcriptional activators in abscisic acid
transcription factors, DREB1 and DREB2, with signaling. Plant Cell 15, 63–78.
an EREBP/AP2 DNA binding domain separate 41) Kazan, K. and Manners, J.M. (2013) MYC2: the
two cellular signal transduction pathways in master in action. Mol. Plant 6, 686–703.
drought- and low-temperature-responsive gene 42) Nakashima, K., Kiyosue, T., Yamaguchi-Shinozaki,
expression, respectively, in Arabidopsis. Plant K. and Shinozaki, K. (1997) A nuclear gene, erd1,
Cell 10, 1391–1406. encoding a chloroplast Clp protease regulatory
31) Stockinger, E.J., Gilmour, S.J. and Thomashow, subunit homolog is not only induced by water
M.F. (1997) Arabidopsis thaliana CBF1 encodes stress but also developmentally up-regulated
an AP2 domain-containing transcription activator during senescence in Arabidopsis thaliana. Plant
that binds to the C-repeat/DRE, a cis-acting J. 12, 851–861.
DNA regulatory element that stimulates tran- 43) Tran, L.S., Nakashima, K., Sakuma, Y., Simpson,
scription in response to low temperature and S.D., Fujita, Y., Maruyama, K. et al. (2004)
water deficit. Proc. Natl. Acad. Sci. U.S.A. 94, Isolation and functional analysis of Arabidopsis
1035–1040. stress-inducible NAC transcription factors that
32) Kasuga, M., Liu, Q., Miura, S., Yamaguchi- bind to a drought-responsive cis-element in the
Shinozaki, K. and Shinozaki, K. (1999) Improving early responsive to dehydration stress 1 promoter.
plant drought, salt, and freezing tolerance by gene Plant Cell 16, 2481–2498.
transfer of a single stress-inducible transcription 44) Fujita, M., Fujita, Y., Maruyama, K., Seki, M.,
factor. Nat. Biotechnol. 17, 287–291. Hiratsu, K., Ohme-Takagi, M. et al. (2004) A
33) Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., dehydration-induced NAC protein, RD26, is
Schabenberger, O. and Thomashow, M.F. (1998) involved in a novel ABA-dependent stress-signal-
Arabidopsis CBF1 overexpression induces cor ing pathway. Plant J. 39, 863–876.
genes and enhances freezing tolerance. Science 45) Tran, L.S., Nakashima, K., Sakuma, Y., Osakabe,
280, 104–106. Y., Qin, F., Simpson, S.D. et al. (2007) Co-
34) Sakuma, Y., Maruyama, K., Osakabe, Y., Qin, F., expression of the stress-inducible zinc finger
Seki, M., Shinozaki, K. et al. (2006) Functional homeodomain ZfHD1 and NAC transcription
analysis of an Arabidopsis transcription factor, factors enhances expression of the ERD1 gene in
DREB2A, involved in drought-responsive gene Arabidopsis. Plant J. 49, 46–63.
expression. Plant Cell 18, 1292–1309. 46) Himmelbach, A., Hoffmann, T., Leube, M.,
35) Shen, Q., Zhang, P. and Ho, T.H. (1996) Modular Hohener, B. and Grill, E. (2002) Homeodomain
nature of abscisic acid (ABA) response complexes: protein ATHB6 is a target of the protein
composite promoter units that are necessary and phosphatase ABI1 and regulates hormone re-
sufficient for ABA induction of gene expression in sponses in Arabidopsis. EMBO J. 21, 3029–3038.
barley. Plant Cell 8, 1107–1119. 47) Urano, K., Maruyama, K., Koyama, T., Gonzalez,
36) Fujita, Y., Fujita, M., Satoh, R., Maruyama, K., N., Inze, D., Yamaguchi-Shinozaki, K. et al.
Parvez, M.M., Seki, M. et al. (2005) AREB1 is a (2022) CIN-like TCP13 is essential for plant
transcription activator of novel ABRE-dependent growth regulation under dehydration stress. Plant
ABA signaling that enhances drought stress Mol. Biol. 108, 257–275.
tolerance in Arabidopsis. Plant Cell 217, 3470– 48) Fujita, M., Mizukado, S., Fujita, Y., Ichikawa, T.,
3488. Nakazawa, M., Seki, M. et al. (2007) Identification
37) Furihata, T., Maruyama, K., Fujita, Y., Umezawa, of stress-tolerance-related transcription-factor
T., Yoshida, R., Shinozaki, K. et al. (2006) genes via mini-scale full-length cDNA over-ex-
Abscisic acid-dependent multisite phosphoryla- pressor (FOX) gene hunting system. Biochem.
tion regulates the activity of a transcription Biophys. Res. Commun. 364, 250–257.
488 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

49) Kim, J.S., Yamaguchi-Shinozaki, K. and Shinozaki, ing twist on the role of ICE1 in Freezing
K. (2018) Er-anchored transcription factors Tolerance. Plant Cell 32, 816–819.
bZIP17 and bZIP28 regulate root elongation. 61) Urano, K., Maruyama, K., Ogata, Y., Morishita, Y.,
Plant Physiol. 176, 2221–2230. Takeda, M., Sakurai, N. et al. (2009) Character-
50) Kim, J.S., Sakamoto, Y., Takahashi, F., Shibata, ization of the ABA-regulated global responses to
M., Urano, K., Matsunaga, S. et al. (2022) dehydration in Arabidopsis by metabolomics.
Arabidopsis TBP-ASSOCIATED FACTOR 12 Plant J. 57, 1065–1078.
ortholog NOBIRO6 controls root elongation with 62) Taji, T., Ohsumi, C., Iuchi, S., Seki, M., Kasuga, M.,
unfolded protein response cofactor activity. Proc. Kobayashi, M. et al. (2002) Important roles of
Natl. Acad. Sci. U.S.A. 119, e2120219119. drought- and cold-inducible genes for galactinol
51) Sakuma, Y., Maruyama, K., Qin, F., Osakabe, Y., synthase in stress tolerance in Arabidopsis thali-
Shinozaki, K. and Yamaguchi-Shinozaki, K. ana. Plant J. 29, 417–426.
(2006) Dual function of an Arabidopsis tran- 63) Nishizawa, A., Yabuta, Y. and Shigeoka, S. (2008)
scription factor DREB2A in water-stress-respon- Galactinol and raffinose constitute a novel func-
sive and heat-stress-responsive gene expression. tion to protect plants from oxidative damage.
Proc. Natl. Acad. Sci. U.S.A. 103, 18822–18827. Plant Physiol. 147, 1251–1263.
52) Sakuma, Y., Maruyama, K., Osakabe, Y., Qin, F., 64) Selvaraj, M.G., Ishizaki, T., Valencia, M., Ogawa,
Seki, M., Shinozaki, K. et al. (2006) Functional S., Dedicova, B., Ogata, T. et al. (2017) Over-
analysis of an Arabidopsis transcription factor, expression of an Arabidopsis thaliana galactinol
DREB2A, involved in drought-responsive gene synthase gene improves drought tolerance in
expression. Plant Cell 18, 1292–1309. transgenic rice and increased grain yield in the
53) Qin, F., Sakuma, Y., Tran, L.S., Maruyama, K., field. Plant Biotechnol. J. 15, 1465–1477.
Kidokoro, S., Fujita, Y. et al. (2008) Arabidopsis 65) Cook, D., Fowler, S., Fiehn, O. and Thomashow,
DREB2A-interacting proteins function as RING M.F. (2004) A prominent role for the CBF cold
E3 ligases and negatively regulate plant drought response pathway in configuring the low temper-
stress-responsive gene expression. Plant Cell 20, ature metabolome of Arabidopsis. Proc. Natl.
1693–1707. Acad. Sci. U.S.A. 101, 15243–15248.
54) Morimoto, K., Ohama, N., Kidokoro, S., Mizoi, J., 66) Urano, K., Maruyama, K., Jikumaru, Y., Kamiya,
Takahashi, F., Todaka, D. et al. (2017) BPM- Y., Yamaguchi-Shinozaki, K. and Shinozaki, K.
CUL3 E3 ligase modulates thermotolerance by (2017) Analysis of plant hormone profiles in
facilitating negative regulatory domain-mediated response to moderate dehydration stress. Plant
degradation of DREB2A in Arabidopsis. Proc. J. 90, 17–36.
Natl. Acad. Sci. U.S.A. 114, E8528–E8536. 67) Mustilli, A.C., Merlot, S., Vavasseur, A., Fenzi,
55) Mizoi, J., Kanazawa, N., Kidokoro, S., Takahashi, F. and Giraudat, J. (2002) Arabidopsis OST1
F., Qin, F., Morimoto, K. et al. (2019) Heat- protein kinase mediates the regulation of stomatal
induced inhibition of phosphorylation of the aperture by abscisic acid and acts upstream of
stress-protective transcription factor DREB2A reactive oxygen species production. Plant Cell 14,
promotes thermotolerance of Arabidopsis thali- 3089–3099.
ana. J. Biol. Chem. 294, 902–917. 68) Yoshida, R., Hobo, T., Ichimura, K., Mizoguchi, T.,
56) Kidokoro, S., Yoneda, K., Takahashi, F., Shinozaki, Takahashi, F., Alonso, J. et al. (2002) ABA-
K. and Yamaguchi-Shinozaki, K. (2017) Different activated SnRK2 protein kinase is required for
cold-signaling pathways function in response to dehydration stress signaling in Arabidopsis. Plant
rapid and gradual decreases in temperature. Plant Cell Physiol. 43, 1473–1483.
Cell 29, 760–774. 69) Yoshida, R., Umezawa, T., Mizoguchi, T.,
57) Kidokoro, S., Hayashi, K., Haraguchi, H., Ishikawa, Takahashi, S., Takahashi, F. and Shinozaki, K.
T., Soma, F., Konoura, I. et al. (2021) Post- (2006) The regulatory domain of SRK2E/OST1/
translational regulation of multiple clock-related SnRK2.6 interacts with ABI1 and integrates
transcription factors triggers cold-inducible gene abscisic acid (ABA) and osmotic stress signals
expression in Arabidopsis. Proc. Natl. Acad. Sci. controlling stomatal closure in Arabidopsis. J.
U.S.A. 118, e2021048118. Biol. Chem. 281, 5310–5318.
58) Kidokoro, S., Kim, J.S., Ishikawa, T., Suzuki, T., 70) Boudsocq, M., Droillard, M.J., Barbier-Brygoo, H.
Shinozaki, K. and Yamaguchi-Shinozaki, K. and Laurière, C. (2007) Different phosphorylation
(2020) DREB1A/CBF3 is repressed by trans- mechanisms are involved in the activation of
gene-induced methylation in the Arabidopsis ice1- sucrose non-fermenting 1 related protein kinases 2
1 mutant. Plant Cell 32, 1035–1048. by osmotic stresses and abscisic acid. Plant Mol.
59) Kim, J.S., Kirokoro, S., Shinozaki, K. and Biol. 63, 491–503.
Yamaguchi-Shinozaki, K. (2020) DNA dimethy- 71) Fujita, Y., Nakashima, K., Yoshida, T., Katagiri,
lase ROS1 prevents inheritable DREB1A/CBF3 T., Kidokoro, S., Kanamori, N. et al. (2009) Three
repression by transgene-induced promoter meth- SnRK2 protein kinases are the main positive
ylation in Arabidopsis ice1-1 mutant. Plant Mol. regulators of abscisic acid signaling in response to
Biol. 104, 575–582. water stress in Arabidopsis. Plant Cell Physiol.
60) Thomashow, M.F. and Torii, K. (2020) SCREAM- 50, 2123–2132.
No. 8] Regulatory gene networks in plant abiotic stress responses 489

72) Nakashima, K., Fujita, Y., Kanamori, N., Katagiri, Takahashi, F., Kidokoro, S. et al. (2017) ABA-
T., Umezawa, T., Kidokoro, S. et al. (2009) Three unresponsive SnRK2 protein kinases regulate
Arabidopsis SnRK2 protein kinases, SRK2D/ mRNA decay under osmotic stress in plants.
SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/ Nat. Plants 3, 16204.
SnRK2.3, involved in ABA signaling are essential 85) Christmann, A., Grill, E. and Huang, J. (2013)
for the control of seed development and dor- Hydraulic signals in long-distance signaling. Curr.
mancy. Plant Cell Physiol. 50, 1345–1363. Opin. Plant Biol. 16, 293–300.
73) Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, 86) Takahashi, F. and Shinozaki, K. (2019) Long-
Y., Christmann, A. et al. (2009) Regulators of distance signaling in plant stress response. Curr.
PP2C phosphatase activity function as abscisic Opin. Plant Biol. 47, 106–111.
acid sensors. Science 324, 1064–1068. 87) Urao, T., Yakubov, B., Satoh, R., Yamaguchi-
74) Park, S.Y., Fung, P., Nishimura, N., Jensen, D.R., Shinozaki, K., Seki, M., Hirayama, T. et al. (1999)
Fujii, H., Zhao, Y. et al. (2009) Abscisic acid A transmembrane hybrid-type histidine kinase in
inhibits type 2C protein phosphatases via the Arabidopsis functions as an osmosensor. Plant
PYR/PYL family of START proteins. Science Cell 11, 1743–1754.
324, 1068–1071. 88) Tran, L.S., Urao, T., Qin, F., Maruyama, K.,
75) Umezawa, T., Sugiyama, N., Mizoguchi, M., Kakimoto, T., Shinozaki, K. et al. (2007) Func-
Hayashi, S., Myouga, F., Yamaguchi-Shinozaki, tional analysis of AHK1/ATHK1 and cytokinin
K. et al. (2009) Type 2C protein phosphatases receptor histidine kinases in response to abscisic
directly regulate abscisic acid-activated protein acid, drought, and salt stress in Arabidopsis. Proc.
kinases in Arabidopsis. Proc. Natl. Acad. Sci. Natl. Acad. Sci. U.S.A. 104, 20623–20628.
U.S.A. 106, 17588–17593. 89) Wohlbach, D.J., Quirino, B.F. and Susman, M.R.
76) Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., (2008) Analysis of the Arabidopsis histidine
Antoni, R., Park, S.Y. et al. (2009) In vitro kinase ATHK1 reveals a connection between
reconstitution of an abscisic acid signaling path- vegetative osmotic stress sensing and seed matu-
way. Nature 462, 660–664. ration. Plant J. 20, 1101–1117.
77) Miyazono, K., Miyakawa, T., Sawano, Y., Kubota, 90) Nishiyama, R., Watanabe, Y., Fujita, Y., Le, D.T.,
K., Kang, H.J., Asano, A. et al. (2009) Structural Kojima, M., Werner, T. et al. (2011) Analysis of
basis of abscisic acid signaling. Nature 462, 609– cytokinin mutants and regulation of cytokinin
614. metabolic genes reveals important regulatory
78) Umezawa, T., Nakashima, K., Miyakawa, T., roles of cytokinins in drought, salt and abscisic
Kuromori, T., Tanokura, M., Shinozaki, K. et al. acid responses, and abscisic acid biosynthesis.
(2010) Molecular basis of the core regulatory Plant Cell 23, 2169–2183.
network in ABA responses: sensing, signaling and 91) Hoang, X.L.T., Prerostova, S., Thu, N.B.A., Thao,
transport. Plant Cell Physiol. 51, 1821–1839. N.P., Vankova, R. and Tran, L.S.P. (2021)
79) Umezawa, T., Sugiyama, N., Takahashi, F., Histidine kinases: Diverse functions in plant
Anderson, J.C., Ishihama, Y., Peck, S.C. et al. development and responses to environmental
(2013) Genetics and phosphoroteomics reveal a conditions. Annu. Rev. Plant Biol. 72, 297–323.
protein phosphorylateion network in the abscisic 92) Nakagawa, Y., Katagiri, T., Shinozaki, K., Qi, Z.,
acid signalling pathway in Arabidopsis thaliana. Tatsumi, H., Furuichi, T. et al. (2007) Arabidop-
Sci. Signal. 6, rs8. sis plasma membrane protein crucial for Ca2D
80) Komatsu, K., Takezawa, D. and Sakata, Y. (2020) influx and touch sensing in roots. Proc. Natl.
Decoding ABA and osmostress signaling in plants Acad. Sci. U.S.A. 104, 3639–3644.
from an evolutionary point of view. Plant Cell 93) Yamanaka, T., Nakagawa, Y., Mori, K., Nakano,
Environ. 43, 2894–2911. M., Imamura, T., Kataoka, H. et al. (2010) MCA1
81) Soma, F., Takahashi, F., Suzuki, T., Shinozaki, K. and MCA2 that mediate Ca2D uptake have
and Yamaguchi-Shinozaki, K. (2020) Plant Raf- distinct and overlapping roles in Arabidopsis.
like kinases regulate the mRNA population up- Plant Physiol. 152, 1284–1296.
stream of ABA-unresponsive SnRK2 kinases 94) Yoshimura, K., Iida, K. and Iida, H. (2021) MCAs
under drought stress. Nat. Commun. 11, 1373. in Arabidopsis are Ca2D-permiable mechanosensi-
82) Takahashi, Y., Zhang, J., Hsu, P.K., Ceciliato, tive channels inherently sensitive to membrane
P.H.O., Zhang, L., Dubeaux, G. et al. (2020) tension. Nat. Commun. 12, 6074.
MAP3Kinase-dependent SnRK2-kinase activa- 95) Nishi, K., Moeller, L.R. and Iida, H. (2021) Mix
tion is required for abscisic acid signal trans- and match: Patchwork domain evolution of land
duction and rapid osmotic stress response. Nat. plant-specific Ca2D-permeable mechanosensitive
Commun. 11, 12. channel MCA. PLoS One 16, e0249735.
83) Katsuta, S., Masuda, G., Bak, H., Shinozawa, A., 96) Yuan, F., Yang, H., Xue, Y., Kong, D., Ye, R., Li,
Kamiyama, Y., Umezawa, T. et al. (2020) C. et al. (2014) OSCA1 mediates osmotic-stress-
Arabidopsis Raf-like kinases act as positive evoked Ca2D increases vital for osmosensing in
regulators of subclass III SnRK2 in osmostress Arabidopsis. Nature 514, 367–371.
signaling. Plant J. 103, 634–644. 97) Choi, W.G., Miller, G., Wallace, I., Harper, J.,
84) Soma, F., Mogami, J., Yoshida, T., Abekura, M., Mittler, R. and Gilroy, S. (2017) Orchestrating
490 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

rapid long-distance signaling in plants with Ca2D, H., Sugimoto, E., Kamiya, A. et al. (2010) ABC
ROS and electrical signals. Plant J. 90, 698–707. transporter AtABCG25 is involved in abscisic
98) Murthy, S.E., Dubin, A., Whitwam, T., Jojoa-Cruz, acid transport and responses. Proc. Natl. Acad.
S., Cahalan, S., Mousavi, S.A.R. et al. (2018) Sci. U.S.A. 107, 2361–2366.
OSCA/TMEM63 are an evolutionarily conserved 112) Kuromori, T., Sugimoto, E. and Shinozaki, K.
family of mechanically activated ion channels. (2014) Intertissue signal transfer of abscisic acid
eLife 7, e41844. from vascular cells to guard cells. Plant Physiol.
99) Hou, C., Tian, W., Kleist, T., He, K., Garcia, V., 164, 1587–1592.
Bai, F. et al. (2014) DUF221 proteins are a family 113) Kuromori, T., Fujita, M., Urano, K., Tanabata, T.,
of osmosensitive calcium-permeable cation chan- Sugimoto, E. and Shinozaki, K. (2016) Over-
nels conserved across eukaryotes. Cell Res. 24, expression of AtABCG25 enhances the abscisic
632–635. acid signal in guard cells and improves plant water
100) Liu, X., Wang, J. and Sun, L. (2018) Structure of use efficiency. Plant Sci. 251, 75–81.
the hyperosmolality-gated calcium-permeable 114) Kang, J., Hwang, J.U., Lee, M., Kim, Y.Y.,
channel OSCA1.2. Nat. Commun. 9, 5060. Assmann, S.M., Martinoia, E. et al. (2010)
101) Wu, F., Chi, Y., Jiang, Z., Xu, Y., Xie, L., Huang, F. PDR-type ABC transporter mediates cellular
et al. (2020) Hydrogen peroxide sensor HPCA1 is uptake of the phytohormone abscisic acid. Proc.
an LRR receptor kinase in Arabidopsis. Nature Natl. Acad. Sci. U.S.A. 107, 2355–2360.
578, 577–581. 115) Kuromori, T., Sugimoto, E. and Shinozaki, K.
102) Kollist, H., Zandalinas, S., Senqupta, S., Nuhkat, (2011) Arabidopsis mutant of AtABCG22, an
M., Kangasjaervi, J. and Mittler, R. (2019) Rapid ABC transporter gene, increase water transpira-
responses to abiotic stress: Priming the landscape tion and drought susceptibility. Plant J. 67, 885–
for the signal transduction network. Trends Plant 894.
Sci. 24, 25–37. 116) Kanno, Y., Hanada, A., Chiba, Y., Ichikawa, T.,
103) Zandalinas, S., Fichman, Y., Devireddy, A., Nakazawa, M., Matsui, M. et al. (2012) Identi-
Sengupta, S., Azad, R.K. and Mittler, R. (2020) fication of an abscisic acid transporter by func-
Systemic signaling during abiotic stress combina- tional screening using the receptor complex as a
tion in plants. Proc. Natl. Acad. Sci. U.S.A. 117, sensor. Proc. Natl. Acad. Sci. U.S.A. 109, 9653–
13810–13820. 9658.
104) Finkelstein, R. (2013) Abscisic acid synthesis and 117) Chiba, Y., Shimizu, T., Miyakawa, S., Kanno, Y.,
response. Arabidopsis Book 11, e0166. Koshiba, T., Kamiya, Y. et al. (2015) Identi-
105) Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., fication of Arabidopsis thaliana NRT1/PTR
Seki, M., Kato, T. et al. (2001) Regulation of FAMILY (NPF) proteins capable of transporting
drought tolerance by gene manipulation of 9-cis- plant hormones. J. Plant Res. 128, 679–686.
epoxycarotenoid dioxygenase, a key enzyme in 118) Shimizu, T., Kanno, Y., Suzuki, H., Watanabe, S.
abscisic acid biosynthesis in Arabidopsis. Plant J. and Seo, M. (2021) Arabidopsis NPF4.6 and
27, 325–333. NPF5.1 control leaf stomatal aperture by regulat-
106) Umezawa, T., Okamoto, M., Kushiro, T., Nambara, ing abscisic acid transport. Genes 12, 885.
E., Oono, Y., Seki, M. et al. (2006) CYP7070A3, a 119) Zhang, H., Zhu, H., Pan, Y., Yu, Y., Luan, S. and
major ABA 8B-hydrooxylase involved in dehydra- Li, L. (2014) A DTX/MATE-type transporter
tion and rehydration response in Arabidopsis facilitates abscisic acid efflux and modulates ABA
thaliana. Plant J. 46, 171–182. sensitivity and drought tolerance in Arabidopsis.
107) Lee, K.H., Piao, H.L., Kim, H.-Y., Choi, S.M., Mol. Plant 7, 1522–1532.
Jiang, F., Hartung, W. et al. (2006) Activation of 120) Takahashi, F. and Shinozaki, K. (2019) Long-
glucosidase via stress-induced polymerization rap- distance signaling in plant stress response. Curr.
idly increases active pools of abscisic acid. Cell Opin. Plant Biol. 47, 106–111.
126, 1109–1120. 121) Takahashi, F., Suzuki, T., Osakabe, Y., Betsuyaku,
108) Endo, A., Sawada, Y., Takahashi, H., Okamoto, M., S., Kondo, Y., Dohmae, N. et al. (2018b) A small
Ikegami, K., Koiwai, H. et al. (2008) Drought peptide modulates stomatal control via abscisic
induction of Arabidopsis 9-cis-epoxycarotenoid acid in long-distance signalling. Nature 556, 235–
dioxygenase occurs in vascular parenchyma cells. 238.
Plant Physiol. 147, 1984–1993. 122) Dhondt, S., Wuyts, N. and Inze, D. (2013) Cell to
109) Sato, H., Takasaki, H., Takahashi, F., Suzuki, T., whole-plant phenotyping: the best is yet to come.
Iuchi, S., Mitsuda, N. et al. (2018) Arabidopsis Trends Plant Sci. 18, 428–439.
thaliana NGATHA1 transcription factor induces 123) Li, D., Quan, C., Song, Z., Li, X., Yu, G., Li, C. et al.
ABA biosynthesis by activating NCED3 gene (2020) High-throughput plant phenotyping plat-
during dehydration stress. Proc. Natl. Acad. Sci. form (HT3P) asa novel for estimating agronomic
U.S.A. 115, 11178–11187. traits from the lab to the field. Front. Bioeng.
110) Kuromori, T., Seo, M. and Shinozaki, K. (2018) Biotch. 8, 62375.
ABA transport and plant water stress responses. 124) Mochida, K., Nishii, R. and Hirayama, T. (2020)
Trends Plant Sci. 23, 513–522. Decoding plant-environment interactions that
111) Kuromori, T., Miyaji, T., Yabuuchi, H., Shimizu, influence crop agronomic traits. Plant Cell Phys-
No. 8] Regulatory gene networks in plant abiotic stress responses 491

iol. 61, 1408–1418. crops. Jpn. Agric. Res. Q. (JARQ) 51, 1–10.
125) Fujita, M., Tanabara, T., Urano, K., Kikuchi, S. 129) Honna, P.T., Fuganti-Pagliarini, R., Ferreira, L.C.,
and Shinozaki, K. (2018) RIPPS: A plant Molinari, M.D.C., Marin, S.R.R., de Oliveira,
phenotyping system for quantitative evaluation M.C.N. et al. (2016) Molecular, physiological,
of growth under controlled environmental stress and agronomical characterization, in greenhouse
conditions. Plant Cell Physiol. 59, 2030–2038. and in field conditions, of soybean plants genet-
126) Kuromori, T., Fujita, M., Urano, K., Tanabata, T., ically modified with AtGolS2 gene for drought
Sugimoto, E. and Shinozaki, K. (2016) Over- tolerance. Mol. Breed. 36, 157.
expression of AtABCG25 enhanced the abscisic 130) Reis, R.R., Cunha, B.A.D.B., Martins, P.K.,
signal in guard cells and improves plant water use Martincs, M.T.B., Alekcevetch, J.C., Chalfan, A.
efficiency. Plant Sci. 251, 75–81. et al. (2014) Induced over-expression of
127) Fernando, A., Selvaraj, M., Ishitani, M., AtDREB2A CA improves drought tolerance in
Nakashima, K., Shinozaki, K. and Yamaguchi- sugarcane. Plant Sci. 221–222, 59–68.
Shinozaki, K. (2021) How utilizing the genes 131) Zandalinas, S.I. and Mittler, R. (2022) Plant
involved in drought tolerance could tackle the responses to multifactorial stress combination.
climate change-related food crisis. Mol. Plant 14, New Phytol. 234, 1161–1167.
1601–1603.
128) Nakashima, K. and Suenaga, K. (2017) Toward the
genetic improvement of drought tolerance in (Received May 24, 2022; accepted July 8, 2022)

Profile

Kazuo Shinozaki was born in Utsunomiya city, Tochigi prefecture, Japan in 1949.
He graduated from Osaka University in 1972. After entering the Graduate Course of
Nagoya University he was awarded his Ph.D., under the supervision of Professors Reiji
and Tsuneko Okazaki, in 1979. His thesis was on discontinuous DNA replication in T7
bacteriophage. He became a Research Associate at the National Institute of Genetics,
Mishima, Japan, in 1978. Then, he became an Assistant Professor in the Department of
Biology, Nagoya University, in 1983, and was promoted to Associate Professor at the
Center for Gene Research, Nagoya University, in 1986. He determined the whole genome
sequence of the tobacco chloroplast with Professor Masahiro Sugiura in 1986. He worked
as a Visiting Scientist with Professor Nam-Hai Chua, Rockefeller University, U.S.A. from
1987 to 1989. He was promoted to Chief Scientist of the Plant Molecular Biology Laboratory of Tsukuba Life
Science Center of RIKEN in 1989. From 1999, he led the plant functional genomics project in the Genome Sciences
Center (GSC) of RIKEN. He became Director of the Plant Science Center (PSC) of RIKEN in 2005, and then
Director of the Center for Sustainable Resource Science (CSRS) of RIKEN in 2013. From 2020, he became Senior
Advisor for RIKEN CSRS. He was also Group Director of the Gene Discovery Research Group of CSRS. He was
President of the Japan Society of Plant Physiologists (JSPP) from 2010 to 2011. He was elected as the University
Professor of Nagoya University in 2017.
Kazuo Shinozaki was awarded the Tsukuba Prize in 2002 and the JSPP Award in 2009. He received a Person
of Merit for Contribution to Culture and Medal with Purple Ribbon for Contribution to Science in Japan in 2016.
He was selected as a corresponding member of the American Society of Plant Biologists in 2015. He was selected as
an international member of the National Academy of Sciences of the U.S.A. in 2020. He was awarded the
International Prize for Biology in 2020 and was awarded the Khalifa International Award for Date Palm and
Agricultural Innovation by the U.A.E. in 2021. From 2014 to 2021, he was selected as a Highly Cited Researcher.
Kazuo Shinozaki has been studying plant regulatory gene networks in abiotic environmental stresses,
especially drought stress. He is known for his studies on the transcriptional regulation of drought stress-inducible
plant genes, signal transduction in drought stress responses and tolerance, and regulatory networks in responses to
the phytohormone abscisic acid. Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki have been collaborating on
these research topics since 1989. Kazuo Shinozaki also contributed to the Arabidopsis functional genomics project
based on the collection of full-length cDNAs and tagged mutant lines for reverse genetics. He is working on
phenotyping of plant growth in various environmental stress conditions.
492 K. SHINOZAKI and K. YAMAGUCHI-SHINOZAKI [Vol. 98,

Profile

Kazuko Yamaguchi-Shinozaki was born in Maebashi city, Gunma prefecture, Japan

in 1954. She graduated from Japan Women’s University in 1977. She entered the
graduate course of Tokyo Institute for Technology and was awarded a Ph.D. under the
supervision of Professor Tsujiaki Hata in 1982. Her thesis was on the translational
regulation of eucaryotic mRNAs. Then, she worked as a postdoctoral fellow at the
National Institute of Genetics under the supervision of Professor Kin-ichiro Miura from
1982. She moved to the Center for Gene Research, Nagoya University, as a special
researcher in 1983. Then, she worked as a postdoctoral fellow in Professor Nam-Hai
Chua’s laboratory from 1987. She worked in RIKEN as a Special Postdoctoral
Researcher from 1989. She became a Chief Researcher at the Japan International
Research Center for Agricultural Research (JIRCAS) in 1993. She was promoted to Professor of the Department of
Agriculture and Life Science, The University of Tokyo in 2004. From 2020, she has been an Emeritus Professor of
the University of Tokyo. She has been Professor of Tokyo University of Agriculture since 2020.
Kazuko Yamaguchi-Shinozaki received the Tokyo Techno Forum Gold Medal in 2000. Then she was awarded
the Tsukuba Prize and the MEXT Minister Award in 2002. She received with the JSPP Award from the Japanese
Society of Plant Physiologists (JSPP) in 2009. She was awarded the MIDORI Academic Prize in 2018 by the Prime
Minister of Japan. From 2014 to 2021, she was selected as a Highly Cited Researcher.
Kazuko Yamaguchi-Shinozaki is a plant molecular biologist mainly working on transcriptional regulatory
networks in environmental stress responses, such as drought, cold, and heat stresses. She discovered key
transcription factors in abiotic stress responses, including abscisic acid responses, and analyzed their upstream
regulatory signaling factors in Arabidopsis. She has applied Arabidopsis and rice genes involved in stress tolerance
for molecular breeding of crops, such as rice, soybean, wheat, sugarcane, and so on based on transgenic technology
through international collaborations. Kazuko Yamaguchi-Shinozaki has been collaborating with Kazuo Shinozaki
on these research topics on plant responses to abiotic stress since 1989.