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.