*2.5. Yeast Strain and Culture Conditions*

The wild type (WT), G19 (Δ*ena1–4*), failure to mediate Na<sup>+</sup> uptake; CY162, the K<sup>+</sup> uptake-deficient, and a yeast strain impaired in spermidine uptake, *agp2*Δ (strains obtained from open biosystems, http://www.openbiosystems.com/GeneExpression/Yeast/ORF/, accessed on 10 May 2022), were used to describe the potential transporter. The yeasts were grown in YPD media at 28 ◦C. The yeast cells were converted using lithium acetate. Put1-8 coding sequences were respectively cloned into the pYES2 expression vector.

Wild type-empty vector and *agp2*Δ-Put1-8 transformants were grown in yeast extract peptone galactose (YPG) medium. Cell suspensions were serially diluted as OD600 of 0.6 for growth tests, and 5 μL aliquots were spotted onto YPG plates containing 25 mM spermidine and 1.5 mM paraquat. After 3–4 days of incubation at 28 ◦C, the plates were photographed. For polyamine transport assays, the yeast cells were harvested at the midlogarithmic phase, washed with ddH2O, and suspended in the YPG media at a dose of 10<sup>8</sup> cells/mL. One-hundred-microliter aliquots cells were transferred to the Eppendorf tubes, and polyamine absorption was activated by the addition of Spermidine or Putrescine at 25 μM concentrations. The absorption was inhibited by adding 1.5 mL of ice-cold uptake

buffer with excessive spermidine content at selected times, filtered by a 0.45 μm membrane, and washed with 2 mL ice-cold ddH2O (three times) to remove the exogenous polyamines. Polyamine determination in vivo was performed by an Agilent high-performance liquid chromatography 1200 series system (HPLC, Agilent Technologies, Santa Clara, CA, USA). Polyamines were obtained from Sigma-Aldrich (St Louis, MO, USA).

For salt tolerance assays, the final pYES2-empty and pYES2-Put1-8 vectors cultured in G19 and CY162 were performed on SD-U (Synthetic Dextrose Minimal Medium without Uracil) medium, and then diluted until the OD600 value = 0.6. 5 μL. Aliquots were spotted onto YPG plates containing 100 mM NaCl and 0.1 mM KCl, respectively, and incubated at 28 ◦C. No treatment was added for the control. After 3–4 days of incubation, the plates were photographed. For Na<sup>+</sup> and K+ uptake treatment, the empty and positive yeast were incubated to OD600 = 1.0, the supporting was discarded, and 50 mL ddH2O was used to wash the yeast. The yeast cells were obtained by centrifugation, followed by starvation treatment with AP liquid medium without K+ and Na+. After starvation treatment, the yeast cells were obtained by centrifugation and treated as follows: inoculating the yeast with an AP liquid medium including 200 μM NaCl and 200 μM KCl, respectively. Subsequently, the liquids were put on a 28 ◦C shaker (220 r/min), and 4 mL of bacterial solution was taken every 10 min, centrifuged, and the supernatant was collected for analysis of Na+ and K+ contents. Three biological replicates were made. The primers for vector construction are listed in Supplemental data S3 (Table S1).
