Figure 1.
Expression of PLDs in response to various stresses.
(A) RNA blotting of PLDα1 and PLDγ transcripts in Arabidopsis leaves exposed to different stress treatments. Arabidopsis Col-0 (3–week old) leaves were detached and floated on water without or with indicated concentrations of chemicals, 1% NaCl, 0.6 M mannitol, 0.2 mM AlCl3, 0.2 mM CdCl2, 2 mM H2O2, 0.1 mM salicylic acid (SA), 0.1 mM methyl salicylate (MeSA), 0.1 mM abscisic acid (ABA), or 0.1 mM methyl jasmonate (MeJA) in a growth chamber at 22°C. Leaves were collected at the indicated times for RNA extraction and northern blotting with [α-32P]-labeled PLDα1 or PLDγ1 full-length cDNA as a probe. (B) RNA blotting of PLDγs using the coding region of PLDγ1 (upper panel) or PLDγ2-specific 5′-UTR (lower panel) under Al stress. Two week-old wild-type seedlings grown in ½ MS medium were treated with 100 µM AlCl3, and roots of seedlings were collected at the indicated time for RNA extraction. [α-32P]-Labeled PLDγ1 cDNA or PLDγ2- specific 5′-UTR was used as a probe for northern blotting. EtBr-stained ribosomal RNA was used as a loading control.
Figure 2.
Generation of PLDγ RNAi and T-DNA insertional knockout mutants.
(A) RNAi suppression construct. Inverted-repeats of an exon (gray boxes) and an intron (empty boxes) of PLDγ1 with restriction sites were cloned in a tandem and expression was driven by double CaMV 35S promoters and terminated by an NOS terminator. (B) Northern blotting analysis of PLDγ1 gene expression in RNAi1 and RNAi2 lines (right panel). The same RNA was probed for PLDα1 expression as a control. EtBr-stained ribosomal RNA was used as a loading control. (C) T-DNA insertion mutant of PLDγ1. Grey boxes show exons and lines between boxes represent introns; T-DNA is inserted at the 8th exon of PLDγ1 (At4g11830, 1912 bp from the start codon of the cDNA). The left border (LB), right border (RB), and direction of T-DNA (arrow in T-DNA) are shown. (D) T-DNA insertion mutant of PLDγ2. T-DNA is inserted at the 6th intron of PLDγ2 (At4g11850, 1635 bp from the start codon of cDNA). (E) Northern blotting of PLDγ1 and PLDγ2 transcripts in mutants and Col-0 using PLDγ1 cDNA and the PLDγ2-specific 5′-UTR of as probes. EtBr-stained 18S RNA is a loading control.
Figure 3.
Root growth of PLDγ mutants and wild-type Arabidopsis under Al stress.
(A-D) PLDγ RNAi mutants and wild-type WS in 1/8 MS medium containing 100 µM AlCl3 (A and B) or in 1/8 MS medium without AlCl3 (C and D) at two different pHs. (E–H) Col-0, pldγ1-1, pldγ2-1 mutants on 1/8 MS plates containing 100 µM AlCl3 (E and F) and 1/8 MS medium without AlCl3 (G and H). Photos are representatives from at least three independent experiments (7 day-old seedlings post treatment).
Figure 4.
Root length and callose accumulation of PLDγ mutants and wild-type roots.
(A) Quantification of root length of PLDγ mutants and wild-type seedlings at various concentrations of AlCl3 (pH 4.0). Four day-old seedlings were transferred to 1/8 MS containing indicated levels of AlCl3. Roots were measured 5 days after transfer. The values of RNAi1 and RNAi2were significantly different (P<0.05) from those of WS and the values for pldγ1-1 were significantly different than the values for Col-0 at 50 and 100 µM AlCl3. Data represent the mean ± SD (N = 50). (B) Callose staining in roots. Seven day-old seedlings were treated with 100 µM AlCl3 in 1/8 MS solution (pH 4.0) for 5 h and roots were stained for callose with anniline blue as described in “Materials and Methods”. Images are representatives of three experiments and more than 25 roots. Control was Col-0 roots without Al treatment; all genotypes had no staining without the Al treatment.
Figure 5.
Al content in roots and organic acid released by roots of PLDγ mutants and wild-type.
Wild-type, pldγ1-1, and pldγ2-1 mutant seedlings were treated in 1/8 MS medium (pH 4.0) containing 50 µM AlCl3 for 5 hrs. Media were used for organic acid analysis with GC-MS (A) and roots were exercised and washed for ICP-MS analysis of Al content (A). Data are from three samples and presented with means ± S.D. * indicates p<0.05, significant difference in Student's t test.
Figure 6.
Changes in oxidative stress and antioxidant activity.
Seven day-old PLDγ mutant and wild type seedlings were treated (white bars) or not treated (black bars) with 100 µM AlCl3 in 1/8 MS solution (pH 4.0) for 5 h or indicated time (for northern blotting). Then roots were harvested for imaging, northern blotting, enzyme activity assay, and lipid peroxidation measurments. (A) ROS generation under Al stress. Roots were stained with 2′,7′-DCFDA for ROS imaging as described in “Materials and Methods”. Images are representatives of three experiments and more than 25 roots. (B) GST expression in plants with altered PLDγ expression under Al stress. RNA extracted from Al-stressed seedling roots for different time was used for northern blotting with [α-32P]-labeled full-length AtGST1 cDNA as the probe. EtBr-stained ribosomal RNA was used as loading controls. (C) Quantification of AtGST1 expression. Quantification was based on band intensity and expressed as ratio to the controls (0 h of treatment). (D) APX and (E) GR activity. Al-treated or control roots were sampled for the enzyme activity assays as described in “Materials and Methods”. (F) Lipid peroxidation. Roots were sampled for assay as described in “Materials and Methods”. Data represent means ± SD (N = 3) from three independent experiments. Label ‘a’ above the bar indicates that the value of the Al-treated is significantly different from the untreated sample of the same genotype at P<0.05. Labels ‘b’ or ‘c’ above the bar indicate that the mutant values are significantly different from wild type with the same treatment at P<0.05.
Figure 7.
Lipid changes as affected by Al stress and PLDγ mutations.
Seven day-old PLDγ mutant and wild type seedlings were treated with (+Al) or without 100 µM AlCl3 (-Al) in 1/8 MS solution (pH 4.0) for 2 days. Roots and shoots (rosette leaves and stems) were harvested separately for lipid extraction as described in “Material and Methods”. Data represent means ± SD (N = 5). (A) and (B) Lipid profiles of PLDγ RNAi mutants and wild-type (WS) seedlings in roots and shoots, respectively. (C) Lipid profiles of PLDγ1-KO and PLDγ2-KO and wild-type (Col-0) seedlings in roots and shoots, respectively. (Label ‘a’ above the bar indicates that the value of the Al-treated is significantly different from that of non-treated control at P<0.05. Label ‘b’ above the bar indicates that the mutant values are significantly different from wild type at P<0.05. Label ‘c’ above the bar indicates that the mutant values are significantly different from wild type and non-Al treatment control at P<0.05.
Figure 8.
Relative changes of glycolipids and phospholipids as affected by Al in roots and shoots.
(A) Ratio of total MGDG/DGDG and glycolipids (GL, sum of total DGDG and MGDG) to phospholipids (PL, sum of total all phospholipids and lysophospholipids) from roots and shoots of PLDγ RNAi mutants and WS seedlings. (B) Ratio of MGDG/DGDG and GL/PL from roots and shoots of Col-0, pldγ1-1, and pldγ2-1 seedlings. The ratios were calculated based on the data in Figure 6. Seven-day-old PLDγ mutant and wild type seedlings were treated with 100 µM AlCl3 (+Al) or without Al (-Al) at pH 4.0 for 2 days. Different letters above the bar indicate sample groups with significant differences (P<0.05) from each other, whereas the same letters indicate no significant difference from each other.