Pdonr221 gateway vector

The stop codon-less cDNA of EX1 and modified EX1 (EX1ΔSOS, EX1W643L, EX1W643A, EX1W565L, EX1F528C, and EX1G646D) were cloned into the pDONR221 Gateway vector (Invitrogen) through the Gateway BP reaction (Invitrogen) and subsequently recombined into the Gateway-compatible plant binary vector pGWB605/pGWB651 for C-terminal fusion with sGFP/G3GFP through the Gateway LR reaction (Invitrogen)^{54 (link)}. An interlace-PCR-based approach was used to prepare amino acid substitution or domain deletion constructs for EX1. The generated vectors were transformed by electroporation into Agrobacterium tumefaciens strain GV3101. Arabidopsis transgenic plants were generated using Agrobacterium-mediated transformation by the floral dip method^{55 (link)}. Transformants were selected on MS medium containing 20 mg/L glufosinate-ammonium (or BASTA; Sigma-Aldrich) until T3 generation to screen for homozygous transgenic plants.

The IPT7::3xGFP plasmid was obtained as follow: 5.8 kb of IPT7 promoter driving nuclear 3xGFP were synthetized by GENEWIZ and inserted into PMLBART vectors via NotI flanking sites.
The SCR::MIR166A plasmid was obtained as follows: chpre-MIR166A was amplified from genomic DNA of Cardamine Oxford ecotype using specific primers: FW 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGGAGGAAGGAAGGGGCTTTCT-3′ REV 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGCCCTAATTAAATTGAGAAGAAGG-3′ and cloned in pDONR221 Gateway vector by BP recombination (Invitrogen). pDONRP4_P1-ChSCRp²⁶ and pDONR221-chpre-miR166A were recombined with pDONR P2R_P3-NOS into a pB7m34GW destination vector via LR reaction (Invitrogen). ChSCRp has been utilized as it is expressed in the endodermis of Arabidopsis thaliana and it is not responsive to salt treatments (Supplementary Fig. 2a and b). Cardamine pre-miR166A (chpre-MIR166A) has been utilized to allow monitoring of endogenous Arabidopsis pre-miR166A response to salt as their sequences partially differ (Supplementary Fig. 2c,²⁶ ). Cardamine miR166A and Arabidopsis miR166A mature forms are identical by sequence²⁶ .

To obtain transgenic rice plants overexpressing the synthetic TvSNAT gene, their full-length sequences were amplified by PCR using specific primers (Table S1) using the synthetic cDNA gene as a template. The initial PCR products were further amplified using a second primer set containing 14 nt of the attB sequence using the initial PCR product as a template. Secondary PCR products were gel purified and cloned into the pDONR221 gateway vector (Invitrogen) via BP recombination. The pDONR221-TvSNAT gene entry vector was then recombined with the pIPKb002 destination vector [29 (link)] via LR recombination to yield the pIPKb002-TvSNAT binary plasmid. Constitutive expression in rice transgenic lines was ensured by the maize ubiquitin promoter.
The binary vector plasmid was transformed into Agrobacterium tumefaciens LBA4404, followed by transformation into rice as described previously [30 (link)].

Full-length rice OsCOMT cDNA (GenBank accession number; AK064768) was kindly provided by the RIKEN BioResource Center (Kikuchi et al., 2003 (link)). Full-length OsCOMT was amplified by PCR using a specific primer set. The forward and reverse primers were 5′-AAA AAG CAG GCT CCA TGG GTT CTA CAG CCG C-3′ and 5′-AGA AAG CTG GGT CTA CTT TGT GAA CTC-3′, respectively. The resulting PCR product was further amplified using a primer set harbouring the attB recombination sequences (forward primer, 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-3′; reverse primer, 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3′). The resulting products were gel-purified and cloned into the pDONR221 Gateway® vector (Invitrogen, Carlsbad, CA, USA) using the BP recombination reaction. The pDONR221:OsCOMT construct was then recombined with the pET300 Gateway destination vector through LR recombination to generate pET300-OsCOMT, followed by transformation into E. coli BL21 (DE3) (Invitrogen). Cell culture and affinity purification steps using a Ni-NTA column were performed according to the manufacturer’s instructions (Qiagen, Tokyo, Japan). Purified recombinant OsCOMT protein was concentrated using an Ultrafree-4 centrifugal filter (Biomax-10, Millipore, Bedford, MA, USA), dissolved in 10mM Tris-HCl (pH 8.0) containing 50% glycerol, and stored at −20 °C until further analysis.

Full-length rice M2H cDNA (AK119413) was amplified by polymerase chain reaction (PCR) using a primer set (forward primer: 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT GCC CGC CGT GGC CGG G-3′; reverse primer: 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT CAG GGT TTG TCG AT-3′), gel-purified, and cloned into the pDONR221 Gateway vector (Invitrogen, Carlsbad, CA, USA) via BP recombination. The resulting pDONR221:M2H entry vector was then recombined with the pK2GW7 Gate destination vector [25 (link)] via LR recombination to form pK2GW7-M2H, which was transformed into Agrobacterium tumefaciens GV2260. Tobacco transformation was conducted according to Duan et al. [26 (link)]. T₁ seeds were screened on Murashige and Skoog (MS) medium containing 200 mg/L kanamycin. After selfing the T₁ plants, T₂ homozygous tobacco lines were selected and used in this experiment.