GoKawiilPlant materials, growth conditions and stress treatment
All the Arabidopsis thaliana mutants and transgenic plants used in this study were in the Columbia (Col-0) background. The T-DNA insertion mutant sam8-1 (SALK_065676) was ordered from the AraShare. The sam8-2 is a CRISPR-edited allele carrying a 1-bp insertion 39 bp downstream of the start codon, which introduces a frameshift and premature stop codon. The resulting protein is MAELQLVEGHQINRRFYPAGDNKLNRSTGNIRRSRSFSRIETIEKT*. The ok130-null mutant was provided by Prof. Pengcheng Wang (Southern University of Science and Technology). Seeds were surface sterilized with 2.5% (v/v) sodium hypochlorite and 70% (v/v) ethanol, stratified for 3 days in the dark at 4 °C, and sown on half-strength Murashige and Skoog (MS), 0.8% (w/v) agar plates supplemented with 1% (w/v) sucrose. Plate media were transferred to a growth chamber under a long-day (16 h light 22 °C/8 h dark 18 °C) photoperiod.
For the germination kinetic assay, all seeds of the indicated genotypes were harvested on the same date from plants grown under identical conditions. They were then uniformly dried at 37 °C for 2 weeks to ensure a consistent after-ripening process. Subsequently, the seeds were sown on the same batch of medium and placed in a growth chamber without stratification under a long-day photoperiod (16 h light at 22 °C/8 h dark at 18 °C). Germination rate was calculated as the mean percentage of seeds that had ruptured their seed coats. Each genotype was evaluated in three biological replicates, each comprising approximately 300 seeds.
For direct osmotic stress treatment, sterilized seeds of different genotypes were germinated on 1/2 MS medium supplemented with 300 mM d-mannitol (Solarbio, M8141) and grown at 22 °C or 26 °C. The phenotypes were recorded at 12 days post-germination. Survival was determined by the ability of seedlings to resume growth after stress removal. Specifically, seedlings were returned to 1/2 MS medium following stress treatment, and survival was assessed 7 days later. Seedlings that remained green and produced new tissues (for example, true leaves) were counted as survivors, whereas those that became white or brown and failed to resume growth were scored as non-survivors. For the transfer assay, sterilized seeds of Col-0 and sam8-1 were germinated on standard half-strength MS medium at 22 °C for 3 days, then transferred to a medium containing 750 mM d-mannitol and grown vertically at 22 °C or 26 °C for 5 days. After treatment, the seedlings were transferred back to normal medium and grown at 22 °C for 10 days before phenotypes were recorded.
For the root extension assay, 5-day-old seedlings grown on standard half-strength MS medium were transferred to a medium containing mannitol and grown vertically for 10 days. For meristem zone measurement, the seedlings were imaged under differential interference contrast of a Nikon AXR with NSPARC confocal microscope system using a 100×/1.45 oil objective 2 days after transfer. The length of the meristem zone was quantified from the QC (Quiescent Center) to the first elongated cell. For the extension rate analysis, root length was measured every day, and the rate was calculated as mm per day.
Plasmid construction
To generate the pSAM8::SAM8-mVenus construct, a 2.2-kb promoter and a 1-kb 3′ untranslated region (UTR) were amplified from wild-type Col-0 genomic DNA and cloned into the pCAMBIA1300-mVenus vector51, giving rise to the pSAM8::mVenus-UTR construct. The coding sequence of SAM8 was amplified and inserted between the promoter and mVenus. For SAM8 variants, site-directed mutagenesis or domain deletion was performed with a standard two-step polymerase chain reaction (PCR) and verified by DNA sequencing to generate the coding sequences of SAM8ΔIDR1, SAM8ΔIDR2, SAM8IDR3mQ, SAM8IDR3mS, SAM8IDR3mK, SAM8IDR3mR, SAM8ΔSAM and SAM8RRKm. All coding sequences were cloned into the pSAM8::mVenus-UTR vector.
To generate the sam8-2 mutant, two sgRNAs were designed and inserted into the BbsI sites of the pAtU6-26-M vector. The Cas9 cassettes were subcloned into pCambia1300-UBQ:Cas9-P2A-GFP-rbcS-E9t vector52 (digested with KpnI and EcoRI). All constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into sam8-1 mutant plants using the standard floral-dipping method. Positive transformants were selected on half-strength MS medium containing 30 mg l−1 hygromycin (AMRESCO, K547). Homozygous transgenic lines were used for experiments. For the sam8-2 mutant, Cas9-free plants were used for experiments.
For the constructs used in transient expression in tobacco epidermal cells, the coding sequences of SAM8 and its variants, ALY1/2/3/4, eIF4A3, UAP56B and NUL1 were amplified and inserted into the pCAMBIA1300-35S-mVenus/NmVenus/CmVenus/Flag/mCerulean/mCherry vector51 (digested with KpnI). The same construct was used for generating overexpression transgenic lines where necessary.
To generate the constructs used for heterologous expression in yeast cells, the coding sequences of SAM8 and its variants were amplified and inserted into the pDUAL-Pnmt1-yeGFP vector53 (digested with NheI and BamHI).
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