PMC

Effect of NaCl on the seed germination phenotype of npc4-1 and npc4-2 knockouts. Forty-five seeds of the WT, npc4-1, or npc4-2 were germinated on an agar plate with or without 150 mM NaCl. Germinated seeds were counted at 24, 30, 36, and 42 h after transferring seeds from 4 °C. Data represent the means ±SE, n=4. This experiment was repeated three times with similar results.

Histochemical analysis of P_NPC4:GUS expression under salt stress in Arabidopsis plants. (A) Effect of salt on the P_NPC4:GUS expression pattern in the main root of 10-day-old seedlings. The plants were grown on agar and transferred to liquid nutrient solution with or without 100 mM NaCl for 24 h. (B) Effect of salt on the P_NPC4:GUS expression pattern in roots of 5-week-old Arabidopsis plants. The plants were grown hydroponically in modified half-strength Hoagland's solution and exposed to 100 mM NaCl for 4 h.

Detailed analysis of NPC4 expression in NaCl-treated plants. The transcript levels of the NPC4 gene were measured by quantitative real-time PCR in leaves and roots of non-treated plants and in plants treated with different concentrations of NaCl (25–100 mM) for different times (1–36 h). Actin2 and UBQ10 were used as internal controls. The expression of NPC4 in non-treated controls at the respective times was set to 1. Data represent the means ±SE, n=2 discrete samples from one biological experiment. This experiment was repeated twice with similar results.

Expression pattern of the NPC genes in NaCl-treated plants. The transcript levels of NPC genes were measured by quantitative real-time PCR in leaves and roots of non-treated plants and in plants treated with 100 mM NaCl for 6 h. Actin2 and UBQ10 were used as internal controls. The expression of each gene in non-treated controls was set to 1. Data represent the means ±SE, n=3 discrete samples from one biological experiment. This experiment was repeated three times with similar results. NPC, non-specific phospholipase C; nd, not determined.

Effect of NaCl on fresh weight and root length of npc4-1 and npc4-2 knockouts. (A) Arabidopsis thaliana Col wild-type (WT), npc4-1, and npc4-2 were grown on agar plates supplemented with 0, 50, and 100 mM NaCl for 14 d. (B) Fresh weight of 14-day-old WT, npc4-1, and npc4-2 seedlings. Forty-five seedlings from one agar plate were pooled and weighed. Data represent the means ±SE, n=4. This experiment was repeated three times with similar results. (C) Root length of 14-day-old WT, npc4-1, and npc4-2 seedlings. Data represent the means ±SE, n=52. This experiment was repeated three times with similar results. Asterisks indicate a statistically significant (t-test P <0.05) difference in comparison with the WT.

Effect of NaCl on DAG production in Arabidopsis seedlings. Arabidopsis seedlings were grown on agar plates for 7 d. Prior to treatment seedlings were removed from agar and incubated with bodipy-PC for 10 min in water. (A) Seedlings were treated for 90 min with 0–100 mM NaCl. Lipids were extracted, separated by high-performance thin-layer chromatography (HP-TLC) and quantified. The quantity of bodipy-DAG in control non-treated seedlings was set to 100%. Data represent means ±SE from independently analysed parallel samples. This experiment was repeated three times with similar results. (B) Seedlings were treated with 100 mM NaCl. Lipids were extracted at the time intervals indicated, separated by HP-TLC, and quantified. The quantity of bodipy-DAG in control non-treated seedlings was set to 100%. Data represent the means ±SE from independently analysed parallel samples. This experiment was repeated twice with similar results. DAG, diacylglycerol.

Relative level of gene expression of ABA-related genes in the WT, npc4-1, and npc4-2 under 100 mM NaCl treatment. The transcript levels of ABI1, ABI2, RAB18, PP2CA, and SOT12 were measured by quantitative real-time PCR in roots of non-treated plants and in plants treated with 100 mM NaCl for 3, 6, 9, and 12 h. Actin2 and UBQ10 were used as internal controls. The expression of the genes in non-treated controls at the respective times was set to 1. Data represent the means ±SE, n=2 discrete samples from one biological experiment. This experiment was repeated three times with similar results.

Relative level of gene expression of SOS signal transduction genes in the WT, npc4-1, and npc4-2 under 100 mM NaCl treatment. The transcript levels of SOS1, CIPK24/SOS2, and CBL4/SOS3 were measured by quantitative real-time PCR in roots of non-treated plants and in plants treated with 100 mM NaCl for 3, 6, 9, and 12 h. Actin2 and UBQ10 were used as internal controls. The expression of the genes in non-treated controls at the respective times was set to 1. Data represent the means ±SE, n=2 discrete samples from one biological experiment. This experiment was repeated three times with similar results.