FIGURE LEGENDS
FIGURE 1 Relationship between the expressions of starch synthesis related genes and CRCT in the leaf sheath. The 8th leaf sheath at 9.5 leaf stage were harvested at early evening. The expressions of starch synthesis related genes and CRCT relative to Actin was analyzed by qRT-PCR and expressed as fold relative to the average value of Nipponbare.
FIGURE 2 Histochemical analysis of starch synthesis related gene promoter::GUS expression. The transgenic lines were grown for 40 days and its tissues were harvested and stained for 2 days. Cross section of leaf blade (a-f) and leaf sheath (g-l) with CRCT (a, g), OsGPT2 (b, h), OsAGPL1 (c, i). OsAGPS1 (d, j).OsBEI (e, k) and OsPho1 (f, l) promoter::GUSconstructs were observed by light microscope. V, vascular bundle; M, mesophyll cell; S, storage parenchyma cell. Scale bar, 50 μm.
FIGURE 3 Diurnal changes in the expression of CRCT. (a) qRT-PCR analysis of CRCT . The expressions of CRCT relative toActin are shown. Data represent mean ± SD of four biological replicates. (b) Western blot analysis of CRCT. The soluble proteins were separated by SDS-PAGE and detected by western blot analysis using anti-CRCT antibody. The arrowhead indicates the position of CRCT. In both experiments, the non-transgenic rice plants were grown in the growth chamber of 14 h light (L) and 12 h dark (D) condition. At indicated time, the leaf blade and leaf sheath were collected.
FIGURE 4 Subcellular localization of CRCT. (a) The chimeric constructs used for GFP assay. The full-length CRCT or CCT domain of CRCT were fused to GFP . Black bar indicates the coding region of CRCT . White box represents the motifs conserved among some CCT proteins. (b) The fluorescence from GFP. The chimeric constructs were introduced into onion epidermal cells. DAPI was used for staining of nucleus. The fluorescence from GFP and DAPI were observed with a fluorescence microscope at 24 h after transformation. Scale bars, 50 μm.
FIGURE 5 ChIP-qPCR analysis of starch synthesis related genes. The 8th leaf sheath at 9.5 leaf stage of FLAG-CRCT expression line was harvested at early evening. ChIP was carried out using anti-FLAG antibodies. Primers used for qPCR were designated at 300-400 bp interval on the 5’ flanking region of starch synthesis related genes (a). Amplicons are shown in red bar. Blue arrowheads indicate positions of CORE element. DNA contents of the promoter regions of OsGPT2 (b),OsAGPL1 (c), OsAGPS1 (d), OsBEI (e) andOsPho1 (f) co-immunoprecipitated with anti-FLAG antibody (ChIP) and in input were analyzed by qPCR. Enrichment of DNAs in ChIP relative to input is shown. Data represent mean ± SD of four biological replicates. 18S ribosome RNA gene was used as a reference for qPCR.
FIGURE 6 Estimation of molecular weight of CRCT by gel filtration chromatography. (a) Immunoblot analysis of the expression of CRCT in the leaf sheaths of CRCT overexpression line and knockout line. Soluble proteins (4.6 μg) were separated by SDS-PAGE, and detected by Coomassie Blue staining (right panel) or immunoblotting using anti-CRCT antibody (left panel). WT, non-transgenic rice; Ox, CRCT overexpression line; crct, CRCT knockout line. (b) Distribution of CRCT in the fractions of gel filtration chromatography. Soluble proteins from leaf sheath of CRCT overexpression line were separated by gel filtration chromatography using the SMART system with a Superose 12 PC3.2/30 column. Proteins in each fractions were separated by SDS-PAGE, and detected by immuno-blotting using anti-CRCT antibody. CR, recombinant CRCT. (c) Estimation of molecular weight of CRCT. Molecular weight standard proteins, IgM pentamer (1048 kDa), apoferritin (720 kDa), Rubisco (550 kDa), B-phycoerythrin (242 kDa), lactate dehydrogenase (146 kDa), bovine serum albumin (66 kDa) and soybean trypsin inhibitor (20 kDa) were also separated by gel filtration chromatography. Molecular weight of CRCT was estimated to be 270 kDa by a linear regression of molecular weight on fraction number. Molecular weight standard proteins and CRCT are indicated as open circle and closed circle, respectively.
FIGURE 7 Immunoprecipitation with FLAG-CRCT. Proteins extracted from the leaf sheathes of CRCT knockout line (crct) and FLAG-CRCT overexpression line (OxFLAG-CRCT) were immunoprecipitated using anti-FLAG antibody (IP). These proteins were separated by SDS-PAGE and detected by immunoblotting using anti-FLAG antibody (left panel) or silver stain (right panel).
FIGURE 8 Yeast two-hybrid assay showing the interaction of CRCT with 14-3-3 proteins. The full-length cDNA of CRCT was cloned into the vector containing the GAL4 activation domain (pAD), and the full length cDNAs of GF14A and GF14B were cloned into the vector containing the GAL4 DNA binding domain (pBD). The transformed yeast were grown on SD2- (/–Leu/–Trp) and SD3-(/–Leu/–Trp/–His) plates. The growths of transformed yeast in SD2- and SD3-indicates the transformation of two vectors and the interaction between two proteins, respectively.
FIGURE 9 Interaction of CRCT with GF14A detected by BiFC assay. N-terminal fusion constructs of CRCT and GF14A with N-terminal (GN) and C-terminal (GC) regions of sfGFP were coexpressed in onion epidermal cells after particle bombardment of the chimeric gene constructs. DsRed protein was also coexpressed as a control for the transformation. Fusion constructs of 7-methylxanthine methyltransferase (MXMT) fromCoffea arabica were used as arbitrary protein control for sfGFP self-assembly. The fluorescence from GFP (green), DsRed (red) and DAPI (blue) was observed with a fluorescence microscope at 24 h after transformation. Scale bars, 50 μm.
TABLE 1 Proteins immunoprecipitated with FLAG-CRCT identified by MS analysis.
RAP-IDa Protein name Molecular MS
mass (kDa) scoreb
Band 1
Os08g0130500 60S acidic ribosomal protein P0 34.5 466
Os01g0686800 Guanine nucleotide-binding protein β 36.7 260
Os01g0896700 60S ribosomal protein L5-2 34.8 153
Band 2
Os04g0462500 14-3-3-like protein GF14B 30.0 906
Os02g0580300 14-3-3-like protein GF14E 29.8 764
Os03g0710800 14-3-3-like protein GF14F 29.3 600
Band 3
Os08g0480800 14-3-3-like protein GF14A 29.1 606
Os08g0430500 14-3-3-like protein GF14C 29.0 434
Proteins showing MS score higher than 200 are listed.
aRAD-ID is the accession number of rice genes in RAP-DB (https://rapdb.dna.affrc.go.jp/).
bMS score is the score assigned by MASCOT search.