SRX12326640 GSM5596408 SRP338570 SRS10299073 GSM5596408 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596408 GSM5596408 GEO Accession GSM5596408 SRX12326641 GSM5596409 SRP338570 SRS10299074 GSM5596409 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596409 GSM5596409 GEO Accession GSM5596409 SRX12326642 GSM5596410 SRP338570 SRS10299072 GSM5596410 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596410 GSM5596410 GEO Accession GSM5596410 SRX12326643 GSM5596411 SRP338570 SRS10299075 GSM5596411 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596411 GSM5596411 GEO Accession GSM5596411 SRX12326644 GSM5596412 SRP338570 SRS10299076 GSM5596412 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596412 GSM5596412 GEO Accession GSM5596412 SRX12326645 GSM5596413 SRP338570 SRS10299078 GSM5596413 OTHER TRANSCRIPTOMIC other A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596413 GSM5596413 GEO Accession GSM5596413 SRX12326646 GSM5596414 SRP338570 SRS10299077 GSM5596414 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596414 GSM5596414 GEO Accession GSM5596414 SRX12326647 GSM5596415 SRP338570 SRS10299079 GSM5596415 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596415 GSM5596415 GEO Accession GSM5596415 SRX12326648 GSM5596416 SRP338570 SRS10299081 GSM5596416 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596416 GSM5596416 GEO Accession GSM5596416 SRX12326649 GSM5596417 SRP338570 SRS10299080 GSM5596417 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596417 GSM5596417 GEO Accession GSM5596417 SRX12326650 GSM5596418 SRP338570 SRS10299082 GSM5596418 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596418 GSM5596418 GEO Accession GSM5596418 SRX12326651 GSM5596419 SRP338570 SRS10299083 GSM5596419 RNA-Seq TRANSCRIPTOMIC cDNA A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction, and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)] in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter, CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C, and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare, IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research, CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina, CA, USA) was used in ¼-scale reactions. For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). NextSeq 500 gds 305596419 GSM5596419 GEO Accession GSM5596419