GoKawiilPlant materials and growth conditions
The hot1 mutant was isolated from an ethyl methanesulfonate-mutagenized M 2 population of the indica cultivar Shuihui527 (R527). Approximately 3,000 M 1 plants were grown in the paddy field, and one M 2 family exhibiting an obviously reduced seed setting rate was identified during the 2017 summer season in Wenjiang, Chengdu, when field temperatures at the heading stage frequently exceeded 35 °C. Heat tolerance assay at the seedling stage (14-day-old plants, 45 °C for 60 h followed by at least 3 days of recovery) confirmed the heat-sensitive phenotype, and the mutant was designated hot1. For subsequent gene mapping, an F 2 population was generated by crossing hot1 with R527.
For functional analyses of OsALA5, several transgenic lines were generated in different genetic backgrounds. A complementation line (OsALA5-C) was produced by introducing the OsALA5 genomic sequence driven by its native 2-kb promoter region into the hot1 mutant background. In the O. sativa cv. Kasalath (Kas) genetic background, two independent knockout lines (OsALA5-KO1 and OsALA5-KO2) were generated using a CRISPR–Cas9 system, and the overexpression lines (OsALA5-OE) were obtained by expressing OsALA5 driven by the CaMV 35S promoter. Additionally, a complementation line (CL) carrying a native promoter-driven OsALA5–GFP fusion construct was created in the OsALA5-KO1 background for subcellular localization and a series of subsequent physiological analyses. All transformation procedures and molecular verifications are described in ‘Generation of transgenic plants’. Unless otherwise stated, rice plants were cultivated in the experimental field of Sichuan Agricultural University (Chengdu, China).
To assess evolutionary conservation of OsALA5, we examined the corresponding Arabidopsis orthologues using both T-DNA and CRISPR–Cas9 mutants. A. thaliana Col-0 served as the wild type. T-DNA insertion mutants37 of ALA9 (ala9, SALK_073953, At1g68710), ALA10 (ala10-2, SALK_024877, At3g25610), ALA11 (ala11-2, SALK_107029, At1g13210) and ALA12 (ala12, SALK_111498, At1g26130) were obtained from the Nottingham Arabidopsis Stock Centre (NASC), and the homozygous lines were verified by PCR-based genotyping following the guidelines provided by the Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org). Double mutants (ala9/12 and ala10-2/11-2) were generated by crossing and verified by PCR. Additional CRISPR–Cas9 knockout lines alleles (ala10-1, ala11-1 and ala10-1/11-1) were produced and confirmed by sequencing. Arabidopsis seeds were surface-sterilized and germinated on 0.5× Murashige and Skoog agar plates at pH 5.8 as described previously51. Ten-day-old seedlings were transferred to soil and grown at 22 °C under long-day conditions (16 h light:8 h dark, 150 µmol m−2 s−1) until harvest. Primers used for vector construction, genotyping and mutation confirmation are listed in Supplementary Table 1.
Heat tolerance assay at the seedling stage
For heat tolerance analysis at the rice seedling stage, healthy seeds were surface-sterilized with 3% sodium hypochlorite for 30 min, rinsed thoroughly with sterile distilled water, and soaked at 37 °C for 3 days to promote uniform germination. Germinated seeds were then placed into bottom-cut 96-well PCR plates and hydroponically cultured in Hoagland’s modified nutrient solution (Coolaber) in a controlled growth chamber (WIPGC-B2P84, BPC600H, Fujian Jiupo Biotechnology) under normal condition (28 °C, 14 h light:10 h dark photoperiod, 150 µmol m−2 s−1 light intensity, and 65% relative humidity). Fourteen-day-old seedlings were transferred to 45 °C for the indicated durations under the same light and humidity conditions and then returned to normal condition for at least three days of recovery.
Seedling survival was evaluated following established criteria52: seedlings that resumed growth and produced new green leaves were scored as alive, whereas those that remained fully bleached and failed to regrow were scored as dead. Survival rates were calculated as the percentage of living seedlings among the total number tested. For the PC spraying assays under heat stress, 14-day-old Kas and OsALA5-KO1 seedlings were prepared as described above. Seedlings were evenly sprayed with 3 ml of buffer control (2% Tween-20 and 2% glycerol), 10 μM PC (18:0/18:0) or 10 μM PC (18:1/18:1). After spraying, seedlings were held under normal growth condition for ~10 min to permit absorption and then subjected to heat treatment at 45 °C for the indicated durations. Survival rates were determined using the same criteria described above. For Arabidopsis heat tolerance assays, 10-day-old seedlings grown on 0.5× Murashige and Skoog agar medium were prepared as described in ‘Plant materials and growth conditions’. Plates were transferred to a 45 °C growth chamber for the indicated durations and then returned to normal condition (22 °C, 16 h light:8 h dark) for at least 3 days of recovery. Survival rates were determined using the same criteria as for rice. For all seedling stage heat tolerance assays, approximately 30 seedlings were grown per pot, and three independent pots were used per genotype for each biological replicate. Each experiment was independently repeated at least three times with consistent results. Data were analysed using GraphPad Prism (v8.0.2). The exact heat treatment durations and conditions for all genotypes and assays are summarized in Supplementary Table 2.
Gene identification
MutMap analysis was performed for gene mapping as previously described16. In brief, an F 2 population of 300 individuals was generated by crossing the hot1 mutant with its wild-type parent R527. Genomic DNA from 30 F 2 individuals displaying the extreme heat-sensitive phenotype was extracted and pooled in equal amounts for whole-genome resequencing (average depth ~25× per sample). Genomic DNA from R527 was resequenced and used as the wild-type reference. The SNP index was calculated for each site to identify genomic regions associated with the mutant phenotype. Candidate SNPs showing complete linkage with the heat-sensitive trait were validated by Sanger sequencing of PCR-amplified genomic fragments. Primers used for SNP validation are listed in Supplementary Table 1.
OsALA5 immunoblotting and immunoprecipitation ATPase assay
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