NLS

1. A 1 25 46 507 aa mTP NLS OsHXK5 GFP OsHXK5-GFP NLS OsHXK5 GFP OsHXK5ΔmTP-GFP mTP OsHXK5 GFP OsHXK5ΔNLS-GFP OsHXK5ΔmTPΔNLS-GFP OsHXK5 GFP OsHXK5NLS-GFP NLS GFP B 506 aa 1 29 45 mTP NLS OsHXK6 GFP OsHXK6-GFP NLS OsHXK6 GFP OsHXK6ΔmTP-GFP mTP OsHXK6 GFP OsHXK6ΔNLS-GFP OsHXK6ΔmTPΔNLS-GFP OsHXK6 GFP OsHXK6NLS-GFP NLS GFP Supplemental Figure S1. Schematic diagrams of all OsHKX-GFP fusion constructs used in subcellular localization experiments. (A) OsHKX5-GFP fusion constructs. (B) OsHKX6-GFP fusion constructs. Mitochondrial targeting peptide sequences (mTPs) and nuclear localization signals (NLSs) are indicated as blue and yellow rectangles.

2. A ZmRbcS::LUC 5 mM 5 mM 0.5 mM 0.5 mM Glc B RAmy3D::LUC Glc Supplemental Figure S2. Expression of glucose responsive genes ZmRbcS (A) and RAmy3D (B) in rice mesophyll protoplasts transfected with the effectors AtHXK1, OsHXK5, OsHXK6, or OsHXK mutant alleles under the control of the CaMV35S promoter in response to glucose treatment. ZmUBQ::GUS was included in each sample as an internal control and control protoplasts were transfected with empty vector. Promoter activities of glucose responsive reporter constructs are represented as relative LUC/GUS activity. All transient expression experiments were repeated three times with similar results.

3. gin2-1 gin2-1 WT WT OsHXK5 OsHXK5 OsHXK6 OsHXK6 - - - - 6% Glucose 6% Mannitol CAB SBP CAA UBQ Supplemental Figure S3. Complementation of the Arabidopsis gin2-1 mutant by expression of OsHXK5 or OsHXK6. (top) Seedlings homozygous for the transgene, and gin2-1 and wild type (WT) seedlings grown on 1/2 MS medium with 6% glucose or 6% mannitol for 6 days. (bottom) Expression levels of CAB, SBP, and CAA measured by RT-PCR analysis in transgenic, gin2-1, and wild type plants. UBQ expression was measured as a control.

4. gin2-1 gin2-1 WT WT OsHXK5 OsHXK5 OsHXK6 OsHXK6 - - - - - - - - OX1 G112D OX1 G113D S186A OX2 S185A OX2 CAB SBP CAA UBQ Supplemental Figure S4. Growth phenotype of transgenic, gin2-1, and wild type (WT) seedling plants grown on glucose-free 1/2 MS medium. (top) Seedlings homozygous for the transgene, and gin2-1 and wild type (WT) seedlings grown on glucose-free 1/2 MS medium. (bottom) Expression levels of CAB, SBP, and CAA measured by RT-PCR analysis in transgenic, gin2-1, and wild type plants. UBQ was used as control.

5. WT gin2-1 OsHXK6 OsHXK5 - - (µmolm-2s-1) 70 240 Supplemental Figure S5. Complementation of the growth defect phenotype of the Arabidopsis gin2-1 by the overexpression of OsHXK5 or OsHXK6in the gin2-1 background. Growth phenotypes of wild type (WT), gin2-1,and transgenic plants under low (70 μmolm-2s-1) (top) or high (240 μmolm-2s-1) light conditions (bottom).