Fig 1.
Interactions between CPK31 and NIP1;1 at the plasma membrane.
(A) Yeast two-hybrid assay of the interactions between CPK31 and NIP1;1. Yeast cells were cotransformed with various combinations of pGBT9- and pGADGH-fusion constructs as indicated on the left of each row. Serial decimal dilutions of corresponding yeast cells were spotted on the synthetic complete agar medium (SC) minus leucine and tryptophan (-Trp-Leu; left lane), SC minus leucine, tryptophan and histidine (-Trp-Leu-His; middle lane), or SC minus leucine, tryptophan, histidine, adenine and contained 40 μM X-α-gal (-Trp-Leu-His-Ade; right lane). Photographs were taken after cultivation at 30°C for 4 days. (B) Subcellular location of CPK31-GFP and NIP1;1-GFP in Arabidopsis cells. The GFP coding sequence was fused to the C-terminus of CPK31 or NIP1;1 coding sequence without stop codon in the pEZS-NL vector, and then transformed into protoplasts of Arabidopsis suspension cultured cells. Empty vector was transformed as a control. To further conform potential plasma membrane location, the protoplasts were stained with the plasma membrane-specific dye FM4-64 (red). (C) CPK31::35S-SPYCE co-expressed with NIP1;1::35S-SPYNE in plasma membrane of Arabidopsis protoplasts observed by bimolecular fluorescence complementation (BiFC). CPK31::35S-SPYCE (CPK31-YC) and NIP1;1::35S-SPYNE (NIP1;1-YN) were transformed into the protoplasts isolated from Arabidopsis suspension cultured mesophylls. The fluorescence signals shown at (B) and (C) were imaged under a Zeiss confocal microscope as green and yellow, respectively (Scale bar: 10 μm).
Fig 2.
Isolation of cpk31 T-DNA insertional mutants and As (III) resistance assays.
(A) Scheme of the Arabidopsis CPK31 gene structure and schematic T-DNA insertion sites of Arabidopsis lines SALK_049228, SALK_007777 and SALK_049236. Solid boxes and lines indicate exons and introns, respectively. The positions of T-DNA insertion in SALK_049228(cpk31-1), SALK_007777(cpk31-2) and SALK_049236 (cpk31-3) are indicated by arrows. (B) RT-PCR analysis of CPK31 and ACTIN2 mRNA levels in wild type (Col-0) and the three mutant lines (cpk31-1, -2, and -3). Three-week-old seedlings grown at half-strength MS were gathered for mRNA concentration analysis. (C) Comparative As(III) sensitivity analysis of wild-type, nip1;1 and cpk31 mutants. The seeds of wild-type (Col-0), nip1;1–1, nip1;1–2, and three cpk31 mutant lines were plated at half-strength MS agar plates containing 0 (a), 5 (b), 10 (c), 20 (d), or 40 μM (e) As(III), and 8-day-old seedlings after germination were used for the photographs. Seedlings of wild-type (Col-0), nip1;1–1, nip1;1–2, and three cpk31 mutant lines were plated on vertical half-strength strength MS agar plates containing 0 (a), 5(b), 10 (c), 20 (d), or 40 μM (e) As(III) for 8 days before taking the photographs. Scale bars = 10 mm. Length of primary roots (D) and whole-plant biomass (E) of wild-type (Col-0), two nip1;1 and three cpk31 mutant lines under various As(III) concentrations. The root length and biomass of 8-day-old seedlings cultured at the conditions described as (C) were measured (n >5 for each condition). Data are mean ± SD of four replicate experiments. Data are mean ± SD of four replicate experiments. Asterisks (*P< 0.05, Student’s t test,) represent statistically significant differences compared with Col-0 as the control.
Fig 3.
Growth phenotype of cpk31 and nip1;1 mutants under variable As(V) conditions.
(A) Growth phenotype of 7-day-old wild-type (Col-0), nip1;1–1, nip1;1–2, cpk31-1, cpk31-2, and cpk31-3 mutants in half-strength MS agar medium containing 0 (a), 150 (b), or 450 μM As (V) (c). Length of primary roots (B) and whole-plant biomass (C) of wild-type (Col-0), two nip1;1 and three cpk31 mutant lines grown for 10 days under 0, 150, or 450 μM As (V). Plants were grown at the normal half-strength MS for 3 days before transferred to the medium containing As(V). Data are mean ± SD of four replicate experiments. It was not statistically significant difference compared with Col-0 as the control (P< 0.05, Student’s t test).
Fig 4.
cpk31-1 and nip1;1–1 mutants contain less As (III) compared with the wild-type.
As (III) contents in shoots (A) and roots (B) of seedlings under As(III) conditions. Three-week-old seedlings of wild-type (Col-0), nip1;1–1 and cpk31-1 mutant grown under the hydroponic medium were transferred to the medium containing 10 μM As (III) for 12, 24, or 48 h. Samples were collected at indicated time points after treatment and As(III) contents were determined. Summarized As(III) content in (A) and in (B) was respectively deduced from results of 5 seedlings/condition of two or four replicate experiments among three independent experiments. Data are mean ± SD, and asterisks indicate statistically significant difference between Col-0 and nip1;1 or cpk31 mutant plants (*P< 0.05, Student’s t test).
Fig 5.
Analysis on tissue expression pattern of CPK31.
(A) Quantification analysis of CPK31 and NIP1;1 transcripts in various organs of the wild-type plants. Total RNA was isolated from various tissues (root, leaf, stem, flower and silique) of 4-week-old wild-type plants (Col-0) grown in soil under long-day conditions. Values were normalized to ACTIN2, and the relative mRNA expression levels were calculated as the ratio of NIP1;1 or CPK31 mRNA level to the NIP1;1 level in rosette leaves of plants (as 1.0). Data are mean ± SD of four replicate experiments. (B-M) Histochemical analysis of CPK31 promoter–GUS expression in transgenic plants. GUS staining in 2-day-old (B), 3-day-old (C), and 7-day-old (D) transgenic seedlings grown on half-strength MS agar plates. The enlarged part of the cotyledon (E), root–hypocotyl junction (F), primary root (G), the tip of primary root (H), and lateral-primary root junction (I) of 7-day-old seedlings. GUS staining in cauline leaf (J), rosette leaf (K), flower (L), and silique (M) of mature CPK31::GUS transgenic plants. To obtain adult plants for staining, 7-day-old seedlings grown on 1/2 MS agar plates were transferred into soil, and samples used for GUS analysis were collected for (J) to (M) on the 21th day after the transfer.
Fig 6.
Expression of CPK31 and NIP1;1 is sensitive to As(III).
The levels of expression of NIP1;1(A) and CPK31(B) in leaves and roots of the seedlings. 3-week-old plants grown at normal hydroponic medium were transferred to the medium with 10 μM As (III), and the roots and shoots of plants incubated for 0, 12, 24, 48, 72, or 144 h were gathered separately for mRNA measurement. After normalized to ACTIN2, the relative expression of NIP1;1 or CPK31 was calculated as the ratio of the normalized expression of NIP1;1 or CPK31 in shoot or root of seedlings treated with As(III) to that in shoot before treated with As(III). Data are mean ± SD of four replicate experiments with n = 3 for each experiment.
Fig 7.
Growth phenotype of cpk31 nip1;1 double mutants under variable As(III) conditions.
(A) qRT-PCR analysis of CPK31, NIP1;1 and ACTIN2 gene expression from the wild-type (Col-0) and cpk31 nip1;1 double mutants (DM). Expression of ACTIN2 was analyzed as a quantitative control. (B) Growth phenotype of 10-day-old wild-type (Col-0), cpk31 nip1;1 double mutants (DM), nip1;1–1, and cpk31-1 mutants grown in half-strength MS agar medium containing 0 (a), 5 (b), 10 (c), 20 (d), or 40μM As(III) (e). The seeds of wild-type (Col-0), cpk31 nip1;1 double mutants (DM), nip1;1–1, and cpk31-1 mutants were firstly planted at the normal half-strength MS agar medium without As(III) for 4 days, and were then transferred for the medium containing various As(III) contents. Length of primary roots (C) and whole-plant biomass (D) of wild-type (Col-0), cpk31 nip1;1 double mutants (DM), nip1;1–1, and cpk31-1 mutants seedlings grown for 10 days under 0, 5, 10, 20, or 40 μM As(III). Data are mean ± SD of four replicate experiments. Asterisks indicate statistically significant difference between Col-0 and mutant plants exposed various contents of As (III) (*P< 0.05, Student’s t test).
Fig 8.
Dexamethasone (DEX)-induced transient CPK31 expression caused a decrease in As(III) tolerance.
(A) Growth phenotype of 7-day-old wild-type (Col-0), cpk31-1, nip1;1–1, cpk31-1 mutant plants transformed with CPK31 expression system (InCPK31) lines 15# (InCPK31#15), 18# (InCPK31#18), and 21# (InCPK31#21) in half-strength MS agar medium containing 0 (a), 2.5 μM As (III) (b), 10 μM DEX (c), or 2.5 μM As (III) and 10 μM DEX (d). (B) Relative expression level of CPK31 in wild-type (Col-0), InCPK31#15, InCPK31#18, and InCPK31#21 lines treated with 10 μM DEX. The seedlings were obtained after 7 day DEX treatment for mRNA measurement. The relative expression of CPK31 was calculated as the ratio of CPK31 in InCPK31 lines to that in the wild type. The data are mean ± SD of three biological replicates. (C) The root length of the wild-type (Col-0), cpk31-1, nip1;1–1, InCPK31#15, InCPK31#18, and InCPK31#21 after 7 days of 10 μM DEX treatment. The data are mean ± SD of three biological replicates.