1. INTRODUCTION
Starch is a major photosynthetic product and is highly accumulated as a
storage polysaccharide in the reproductive organs of plants such as
seeds and tubers. In addition, the starch is utilized for transient
carbon storage of photosynthate in vegetative organs, i.e., leaf for
most plants as well as leaf sheath for some gramineous plants, including
rice. Rice is an important food crop and a model plant for the genomic
analysis of monocots. In rice, starch is highly accumulated in the leaf
sheath during the vegetative stage and remobilized to the reproductive
organs at the grain-filling stage. This carbon remobilization accounted
for no less than 30% of grain carbon (Cook & Yoshida, 1972), which can
be particularly important for securing yield under limited solar
radiation during the grain-filling stage (Okamura et al., 2013).
A greater capacity of starch accumulation in the leaf sheath can reduce
the occurrence of chalky grains under elevated temperature (Morita &
Nakano, 2011). Thus, the capacity of starch accumulation in the
vegetative organ is an important determinant of rice yield and quality.
Starch synthesis is a complex biochemical reaction catalyzed by multiple
enzymes such as ADP-glucose pyrophosphorylase (AGPase), starch synthase,
granule-bound starch synthase, α-glucan phosphorylase (Pho), starch
branching enzyme (BE) and starch debranching enzyme. It is well known
that the starch level increases markedly in vegetative organs of plants
when grown under elevated CO2 conditions (Ainsworth, &
Long, 2005). Previously, we screened CO2-responsive
genes by microarray and identified a gene encoding a novel CONSTANS,
CONSTANS-Like and TOC1 (CCT) domain-containing protein, designatedCO2-responsive CCT protein (CRCT ), in rice
(Fukayama et al., 2009; Morita et al., 2015). The starch
content in the leaf sheath was greatly increased in CRCToverexpression lines and decreased in CRCT knockdown lines
(Morita et al., 2015). The expression of multiple genes related
to starch synthesis such as AGPase large subunit 1(OsAGPL1 ) and BE I (OsBEI ) were highly correlated
with the expression levels of CRCT in those transgenic rice
lines, suggesting that CRCT is a positive regulator of starch
accumulation in vegetative tissues. In addition, the increase in
capacity of starch accumulation in vegetative organs by CRCToverexpression led to the enhancement of photosynthetic capacity of rice
when grown under elevated CO2 condition (Morita et
al., 2016). Moreover, analysis of the chain-length distribution
of starch showed that the level of short chains with a degree of
polymerization from 5 to 14 was positively correlated with the
expression level of CRCT (Morita et al., 2019). These
results suggest that CRCT can control the structure as well as
the quantity of starch in the vegetative organs.
CRCT contains a CCT domain. In plants, CCT domain-containing proteins
are mostly involved in the processes of photoperiodic flowering or
circadian rhythms (Valverde, 2011; Li, & Xu, 2017). CCT
domain-containing proteins are divided into three groups: the
CONSTANS-like (COL) family with one or two zinc-finger B-box domains,
the pseudo-response regulator family with a pseudo receiver domain and
the CCT motif family with no such additional conserved domains (Cockram
et al., 2012). CRCT belongs to the CCT motif family (Morita et
al., 2015), and this family is thought to have evolved from a common
ancestor COL gene and lost its B-box domains (Cockram et al.,
2012). Although there have been no reports of functional analysis ofCRCT orthologue in other plants, CCT motif family members are
known to have diverse physiological functions in plants. For example,Grain number, plant height, and heading date 7 (Ghd7 ) is a
pleiotropic gene that controls the plant height, chlorophyll content,
and yield, as well as the heading date in rice (Xue et al., 2008; Wang
et al., 2015). ZmCCT was reported to be a major determinant of
tassel size in maize (Xu et al., 2017). ASML2 inArabidopsis thaliana responds to sugar level and regulates some
carbon metabolism-related genes (Masaki et al., 2005). Considering these
functions, CCT motif family genes may play an important role in
determining the plant growth and yield in plants.
The CCT domain has a nuclear localization signal (NLS) and thus is
responsible for nuclear localization of proteins with this domain
(Robson et al., 2001). More importantly, the CCT domains of CONSTANS and
TOC1 were found to bind directly to DNA with CORE elements (Tiwari et
al., 2010; Gendron et al., 2012). In addition, CONSTANS physically
interacts with Nuclear Factor-Y transcription factors (NF-YB and NF-YC)
to bind CORE elements in Arabidopsis (Gnesutta et al., 2017).
Heading date 1, an orthologue of CONSTANS in rice, interacts with Ghd7
and binds to the promoter of a key flowering inducer, Early heading date
1 (Nemoto et al., 2016). The founding member of a PRR family CCT
protein, TOC1 can interact with the F-box protein ZEITLUPE to control
the circadian clock genes (Fujiwara et al., 2008). Thus, these
representative CCT proteins function by interacting with other proteins.
CRCT can be described as a positive regulator of starch synthesis but
the molecular function of this protein has been largely unknown. In this
study, we carried out chromatin immunoprecipitation (ChIP) assays and
showed that CRCT can bind to the promoter region of starch synthesis
related gens. In addition, we used immunoprecipitation to identify
14-3-3 proteins associated with CRCT. This interaction was confirmed by
yeast two-hybrid analysis and bimolecular fluorescence complementation
(BiFC) assays. These findings gave us clues to a possible mechanism of
how CRCT controls starch synthesis in vegetative organs.