PMC

Phenotypes of salt-tolerant accessions using the mesh-transferred assay. Seven-day-old Bu-5, Col-0, Ll-1, Bur-0, Wl-0, and Zu-0 seedlings grown on a nylon mesh on MS agar plates were mesh transferred to plates supplemented with 250 mM NaCl for 7 d.

Expression of SA-inducible genes in Col-0 and Bu-5. Total RNA (10 μg per lane) was isolated from 7-day-old Col-0 and Bu-5 seedlings subjected to 100 mM NaCl as SA. The northern blots were sequentially probed with the indicated probes belonging to Groups I–III of the microarray analyses.

Evaluation of stress tolerance of sorbitol, CsCl, and LiCl after SA of salt-tolerant accessions. (A) Salt-acclimated 2-week-old Col-0 and Bu-5 seedlings were mesh transferred to MS agar plates containing 750 mM sorbitol, 5 mM CsCl, or 25 mM LiCl. (B) Salt-acclimated Bu-5, Col-0, Ll-1, Bur-0, Wl-0, and Zu-0 plants were exposed to 750 mM sorbitol for 14 d.

Salt tolerance after salt acclimation (SA) of Col-0 and Bu-5 plants. (A) Flowchart of the SA assay. The arrow shows a mesh transfer. (B) Phenotypes of Col-0 and Bu-5 seedlings with and without SA under 300 mM NaCl. (C) Chlorophyll content in shoots under 30 mM NaCl for 7 d with and without SA. The contents were assessed based on fresh weight (FW). Values are the mean ±SE; n=5.

Weekly photographs of salt-tolerant or salt-sensitive accessions under salinity stress using the soil pot assay. Three-week-old Bch-4, Cvi-0, Sg-2, Sh-0, Col-0, Bu-5, Bur-0, Ll-1, and Zu-0 plants grown in soil pots were exposed to 500 mM NaCl in water for 35 d. It shows the weekly photographs from day 0 to the 35th day after salt stress treatment.

Frequency distribution of days of survival of 344 Arabidopsis thaliana accessions under salt stress using soil pots. Three-week-old plants grown in soil pots were exposed to 500 mM NaCl in water. Survival days represent the number of days from the initiation of stress until complete chlorosis was observed in all plants in a pot. Arrows indicate the average (n=20) positions corresponding to a reference accession Col-0, and markedly salt-tolerant accessions including Bu-5, Bur-0, Ll-1, Wl-0, and Zu-0, respectively.

Na⁺ and K⁺ contents in Col-0 and Bu-5 during the SA assay. (A) Na⁺ contents in rosette leaves of Col-0 and Bu-5. (B) K⁺ contents in rosette leaves of Col-0 and Bu-5. Seven-day-old Col-0 and Bu-5 seedlings grown on nylon mesh on MS agar plates were mesh transferred to plates with 100 mM NaCl for 7 d as SA and subsequently mesh transferred to plates with 300 mM NaCl for 3 d. The dashed line shows the transfer point from 100 mM to 300 mM NaCl. Na⁺ and K⁺ contents were based on fresh weight (FW). Values are mean ±SE; n=5. Differences between Bu-5 and Col-0 were analysed by Student's t-test. *P <0.05; ***P <0.001.

Classification of SA-inducible genes in Col-0 and Bu-5. In total, 213 and 142 SA-inducible genes of Col-0 and Bu-5, respectively, were identified by microarray analyses. In addition, 271 genes expressed strongly in Bu-5 as compared with Col-0 on day 7 under SA were identified. These genes were categorized into seven groups: Group I, genes induced in Bu-5 and expressed strongly in salt-acclimated Bu-5 compared with salt-acclimated Col-0; Group II, genes induced in both Col-0 and Bu-5 and expressed strongly in salt-acclimated Bu-5 compared with salt-acclimated Col-0; Group III, genes induced in Col-0 and expressed strongly in salt-acclimated Bu-5 compared with salt-acclimated Col-0; Group IV, genes expressed strongly in salt-acclimated Bu-5 compared with salt-acclimated Col-0; Group V, genes induced in Bu-5; Group VI, genes induced in Col-0; Group VII, genes induced in both Col-0 and Bu-5. The number of SA-inducible genes whose expression ratios are >3-fold is indicated. In addition, the number of genes expressed 1.5 times more highly in Bu-5 than in Col-0 on day 7 under SA is indicated. Fold change was calculated from the normalized score based on the signal intensity.

Genetic architecture of NaCl tolerance without SA. (A) Salt tolerance of F₁ progeny derived from crosses between Bur-0 and Col-0 plants. Two-week-old seedlings of Bur-0, Col-0, and their F₁ progeny grown on a nylon mesh on MS agar plates were mesh transferred to plates with 225 mM NaCl for 7 d. (B) F₂ segregation analysis. Two-week-old Bur-0, Col-0, and their F₁ progeny grown on a nylon mesh on MS agar plates were mesh transferred to plates with 225 mM NaCl for 7 d. Arrowheads indicate F₂ seedlings with the recessive phenotype. (C) Genetic mapping of loci controlling the differential response to salt stress without SA. Recombination frequency derives from the salt-sensitive F₂ population. The scores indicate recombination frequencies (%). n=44. SSLP markers are described in Supplementary Table S2 available at JXB online. Dots represent centromeres.

Genetic architecture of osmotic stress tolerance after SA. (A) Salt tolerance of F₁ progeny derived from crosses between Bu-5 and Col-0 plants. Two-week-old seedlings of Bu-5, Col-0, and their F₁ progeny grown on a nylon mesh on MS agar plates were mesh transferred to plates with 100 mM NaCl (SA) and subsequently transferred to plates with 750 mM sorbitol for 14 d. (B) F₂ segregation analysis. Salt-acclimated Bu-5, Col-0, and their F₁ progeny were mesh transferred to MS agar plates with 750 mM sorbitol for 14 d. Arrowheads indicate F₂ seedlings with the recessive phenotype. (C) Genetic mapping of loci controlling the differential response to osmotic stress after SA. The scores indicate recombination frequencies (%), which are derived from osmosensitive F₂ populations [Col-0×Bu-5 (n=42–44), Col-0×Bur-0 (n=20), Col-0×Cal-0 (n=20), Col-0×Ll-1 (n=18), and Col-0×Zu-0 (n=20)]. nd indicates no polymorphism between Col-0 and the accession when the SSLP marker was used. The SSLP markers are described in Supplementary Table S3 at JXB online. Dots represent centromeres.