A 25 NLS OsHXK5-GFP OsHXK5ΔmTP-GFP OsHXK5ΔNLS-GFP OsHXK5ΔmTPΔNLS-GFP OsHXK5NLS-GFP 46 507 aa 1 OsHXK5 GFP mTP B 29 NLS OsHXK6-GFP OsHXK6ΔmTP-GFP OsHXK6ΔNLS-GFP OsHXK6ΔmTPΔNLS-GFP OsHXK6NLS-GFP 45 506 aa 1 OsHXK6 GFP mTP 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.

A ZmRbcS::LUC Vector AtHXK1 OsHXK5 OsHXK5-G113D OsHXK5-S186A OsHXK6 OsHXK6-G112D OsHXK6-S185A Glc 5 mM 0.5 mM B RAmy3D::LUC Vector AtHXK1 OsHXK5 OsHXK5-G113D OsHXK5-S186A OsHXK6 OsHXK6-G112D OsHXK6-S185A Glc 5 mM 0.5 mM 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.

CAB SBP CAA UBQ 6% Mannitol 6% Glucose WT OsHXK5 OsHXK6 gin2-1 - 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.

- WT OsHXK5 OsHXK6 gin2-1 OX1 OX2 - WT OsHXK5 OsHXK6 gin2-1 G113D G112D S185A S186A 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.

WT gin2-1 - - OsHXK5 OsHXK6 (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 OsHXK6 in 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).