Arabidopsis PLDzeta2 regulates vesicle trafficking and is required for auxin response

Abstract

Phospholipase D (PLD) and its product, phosphatidic acid (PA), play key roles in cellular processes, including stress and hormonal responses, vesicle trafficking, and cytoskeletal rearrangements. We isolated and functionally characterized Arabidopsis thaliana PLDzeta2, which is expressed in various tissues and enhanced by auxin. A PLDzeta2-defective mutant, pldzeta2, and transgenic plants deficient in PLDzeta2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29 degrees C, whereas transgenic seedlings overexpressing PLDzeta2 showed opposite phenotypes, suggesting that PLDzeta2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the beta-glucuronidase (GUS) expression in crosses between pldzeta2 and lines containing DR5-GUS indicated that PLDzeta2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLDzeta2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLDzeta2 overexpression. Analyses of crosses between pldzeta2 and lines containing PIN-FORMED2 (PIN2)-enhanced green fluorescent protein showed that PLDzeta2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLDzeta2 and PA are required for the normal cycling of PIN2-containing vesicles as well as for function in auxin transport and distribution, and hence auxin responses.

Figure 1.

Structural Organization and Expression Pattern Analysis of PLDζ2. (A) Diagram illustrating conserved domains and motifs present in the deduced protein of PLDζ2. AA, amino acid; PX, Phox-homology domain; PH, Pleckstrin-homology domain; HKD, H(X)K(X)(4)D conserved hydrolysis domain; DRY, Gα-interacting motif. (B) Expression pattern analysis of PLDζ2. RT-PCR analysis revealed the expression of PLDζ2 in multiple tissues (top). Arabidopsis Actin2 was used as an internal positive control. Promoter-GUS fusion experiments (bottom) indicated PLDζ2 expression in seedlings, especially roots and cotyledons (panels 1 to 4, arrows), the elongation zone of primary roots (panels 1 to 3 and 5), guard cells of cotyledons (panel 6), anthers (panel 7), pollen grains (panel 8), and immature seeds (panel 9). PLDζ2 was also expressed at different stages of embryo development, including the early stage after fertilization (panel 10), the globular stage (panel 11), and the mature cotyledon stage (panel 12). Arrows highlight GUS signals. Bars = 200 μm (panels 1 and 5), 20 μm (panels 6 and 8), 500 μm (panels 2 to 4, 7, and 9), and 100 μm (panels 10 to 12). (C) PLDζ2 expression was enhanced by IAA. RT-PCR analysis showed that PLDζ2 expression was stimulated by 100 μM IAA treatment for various times (left panel; 7-d-old seedlings were treated). Actin2 was used as an internal positive control. Analysis of transgenic Arabidopsis plants carrying the PLDζ2 promoter-GUS fusion construct revealed that PLDζ2 expression in roots was enhanced by external IAA application (right panel; 7-d-old seedlings were treated with 1 μM IAA for 48 h). Bar = 200 μm.

Figure 2.

The Knockout Mutant pldζ2 Showed Reduced Responses to Auxin. (A) Diagram showing the positions of the T-DNA insertion in the PLDζ2 genomic sequence and primers used to identify the knockout mutant pldζ2. Primers P2 and LBa1, P1 and P2, and P3 and P4 were used to confirm the presence of T-DNA, to screen the homozygous line, and to detect the transcripts of PLDζ2, respectively (top). RT-PCR analysis revealed the deficiency of PLDζ2 transcripts in homozygous mutant plants. Actin2 was used as an internal positive control (bottom). (B) Measurement and statistical analysis of relative root growth indicated the reduced auxin responses of pldζ2 seedlings. The auxin-insensitive mutant tir1-1 was used as a positive control. Nine-day-old seedlings were used for observation and measurement. Error bars represent se (n > 30), and statistical analysis was performed using a one-tailed Student's t test (* significant difference at P < 0.01). (C) pldζ2 seedlings showed repressed gravity responses compared with wild-type controls. Five-day-old vertically grown seedlings were reoriented by 90°, and curvatures were measured at different times after reorientation. Error bars represent se (n > 60), and a one-tailed Student's t test indicated significant differences (*P < 0.01). (D) pldζ2 seedlings were less sensitive to auxin-dependent hypocotyl elongation under high temperature (left). Hypocotyl lengths of 7-d-old seedlings grown at 22 or 29°C were measured and statistically analyzed (right). The auxin-insensitive mutants tir1-1 and axr1-3 were used as positive controls. Bar = 2 mm. Error bars represent se (n > 30), and a one-tailed Student's t test indicated significant differences (*P < 0.01).

Figure 3.

Modified Expression of PLDζ2 Resulted in Altered Sensitivity to Auxin. (A) Enhanced expression of PLDζ2 in transgenic lines (LO2, LO11, and LO19; left) prepared by genetic transformation using construct pO-PLDζ2. Suppressed expression of PLDζ2 in transgenic lines (LA3, LA8, LA9, and LA11; right) developed by genetic transformation using construct pA-PLDζ2. Total RNA samples were extracted from 7-d-old seedlings. PLDζ2 transcript levels were analyzed using RT-PCR, and Actin2 was used as an internal control. C, control plants. (B) Measurement and statistical analysis of relative root elongation of seedlings with altered PLDζ2 expression grown on medium supplemented with various concentrations of IAA for 9 d confirmed the hypersensitivity to auxin of PLDζ2-overexpressing plants and the relative insensitivity of PLDζ2-deficient plants. Primary root lengths of untreated plants were set as 100%. Error bars indicate se (n > 40), and statistical analysis was performed using a one-tailed Student's t test (*P < 0.01). (C) Time course of curvature in seedling gravity response tests. Curvatures of 5-d-old control and transgenic plants with enhanced or decreased PLDζ2 expression levels were measured after 90° reorientation and analyzed statistically (n > 40). Error bars represent se, and statistical analysis was performed using a one-tailed Student's t test (*P < 0.01).

Figure 4.

PA Stimulated Root Gravity Responses. (A) Effects of PA on root gravity response. Five-day-old Arabidopsis seedlings were transferred onto medium supplemented with or without 10 μM PA, and seedling curvatures were measured and analyzed statistically (n > 40) as described for Figure 3C. Error bars represent se. * significant difference at P < 0.01; ^# significant difference at P < 0.05. (B) Effects of the PLD-specific inhibitor 1-butanol on root gravity response. Five-day-old seedlings were transferred onto medium supplemented with or without 1-butanol (0.2 or 0.4%, v/v), 2-butanol (0.4%, v/v), or 3-butanol (0.4%, v/v), and seedling curvatures were measured and analyzed statistically (n > 60). Results showed that 1-butanol treatment severely suppressed seedling gravity responses, whereas no obvious changes were found with 2-butanol or 3-butanol treatment. Error bars represent se.

Figure 5.

Effects of PLDζ2, PA, and the PLD-Specific Inhibitor 1-Butanol on Auxin Distribution and Response, Evaluated by Detecting DR5-GUS Expression. For all analyses, the GUS signals shown are representative of >20 independent samples. (A) PLDζ2 positively regulates DR5-GUS expression. Five-day-old seedlings were used, and GUS signals were examined in cotyledons (top) and roots (bottom). Left panel, wild-type control; left center panel, pldζ2; right center and right panels, PLDζ2-overexpressing lines. Bar = 500 μm (top) and 50 μm (bottom). (B) PA stimulates DR5-GUS expression. Dose concentrations of PA (10, 20, or 50 μM) were supplemented and applied for 24 or 48 h. Roots of 5-d-old seedlings were observed. Bar = 100 μm. (C) 1-Butanol treatment suppresses DR5-GUS expression. GUS activities were detected under treatment with 1-butanol or 2-butanol (0.4 or 0.8%, v/v) for 24 h (top) or 48 h (bottom). Bar = 100 μm. (D) 1-Butanol suppresses auxin-induced DR5-GUS expression in the root elongation zone. Seedlings harboring the DR5-GUS construct were treated with 10 μM IAA and different concentrations of 1-butanol (0.2, 0.4, 0.6, or 0.8%, v/v) or 2-butanol (0.8%, v/v) for 3 h. Bar = 50 μm.

Figure 6.

Expression of Auxin-Responsive Genes Was Modulated under Altered Expression of PLDζ2. Relative expression levels of three early auxin-responsive genes, IAA5 (A), IAA19 (B), and GH3-3 (C), were detected in wild-type control, pldζ2, and transgenic plants deficient in or overexpressing PLDζ2 (pA-PLDζ2-LA11, pO-PLDζ2-LO11, and LO19, respectively) in the absence or presence of IAA by quantitative real-time RT-PCR analysis. Induction of three genes by IAA was suppressed in pldζ2 and transgenic plants deficient in PLDζ2, whereas it was enhanced in PLDζ2-overexpressing plants. Seven-day-old seedlings were treated with 10 μM IAA for 90 min. Gene transcript abundance was analyzed, and relative intensity was calculated. The experiments were repeated three times, and the data shown are means plus se. The amplification of Actin7 was used as a control.

Figure 7.

PLDζ2 Stimulated Vesicle Trafficking. (A) Compared with the wild-type control (panels 1 and 6), deficiency of PLDζ2 suppressed the internalization of FM4-64 (panels 2, 5, 7, and 10), indicating decreased vesicle trafficking. Alternatively, internalization of FM4-64 was clearly stimulated in PLDζ2-overexpressing plants (panels 3, 4, 8, and 9). Roots of 7-d-old seedlings (n > 15) were stained with the membrane-selective dye FM4-64 (5 μM, 10 min) and then observed after incubation for 20 or 45 min. Arrowheads highlight the vesicles labeled with FM4-64. Bars = 5 μm. (B) Compared with the untreated control (panel 1), 1-butanol decreased FM4-64 internalization (panels 2, 3, 5, and 6), whereas PA enhanced it (panel 4). Roots of 7-d-old seedling (n > 15) were treated with 1-butanol (0.4 or 0.8%, v/v) for 3 h (panels 2 and 3) or 12 h (panels 5 and 6) or with PA (50 μM, 3 h; panel 4), followed by staining with FM4-64 (5 μM, 10 min), and observed after incubation for 45 min. Arrowheads highlight the vesicles labeled with FM4-64. Bars = 5 μm.

Figure 8.

PLDζ2 Suppressed the Inhibitory Effects of BFA on Vesicle Trafficking. (A) Compared with the wild-type control (panel 1), BFA compartments were detected in only a few cells of PLDζ2-overexpressing roots (panels 2 and 3). However, the number of root cells containing BFA compartments was largely indistinguishable between PLDζ2-deficient plants (panels 4 to 6) and the wild-type control (panel 1). Roots of 7-d-old seedlings were first treated with BFA (50 μM) for 2 h, then incubated with 50 μM BFA and 5 μM FM4-64 for 30 min, followed by examination by confocal microscopy. Arrowheads highlight BFA compartments labeled with FM4-64. Bars = 10 μm. For all analyses, the results shown are representative of >15 independent samples. (B) Compared with the wild-type control and pldζ2, PLDζ2-overexpressing plants (LO11 and LO19) were insensitive to BFA inhibition of primary root elongation (left) and lateral root formation (right). Five-day-old seedlings were transferred onto medium supplemented with (5 μM) or without BFA for another 7 d, and primary root lengths and lateral root numbers were determined and analyzed statistically. Error bars represent se (n > 40). * significant difference (P < 0.01). (C) Compared with control and pldζ2 seedlings, PLDζ2-overexpressing plants (LO11 and LO19) had reduced sensitivity to BFA inhibition of the root gravity response. Five-day-old seedlings were transferred onto medium supplemented with (bottom row; 10 μM) or without (top row) BFA, and root curvatures were measured after 135° reorientation and growth for 36 h. The curvature of each root was assigned to one of 12 30° sectors (n > 40).

Figure 9.

PLDζ2, 1-Butanol, and PA Suppressed BFA Inhibition on PIN2 Cycling. The PIN2-EGFP cassette was individually transferred into pldζ2 or PLDζ2-overexpression (LO11 and LO19) backgrounds through genetic crosses. The resultant progeny were used in this series of experiments. (A) In the absence of BFA, polar localization patterns of PIN2 (panels 1 to 4) were indistinguishable in wild-type, pldζ2, or PLDζ2-overexpression backgrounds, indicating that manipulation of PLDζ2 expression levels does not affect the polar localization of PIN2. After BFA treatment (50 μM, 2 h), accumulation of PIN2 in the PLDζ2-overexpression background (panels 7 and 8) was much less than that in the wild type (panel 5) and pldζ2 (panel 6). After BFA washout for 2 h, resumption of PIN2 polar localization was more complete in the PLDζ2-overexpression background (panels 11 and 12) than in the wild-type (panel 9) and pldζ2 (panel 10). For all analyses, roots of 5-d-old progeny were used (n > 15). Bars = 10 μm. (B) Treatment with 1-butanol, 2-butanol, or PA did not affect the polar localization of PIN2 (panels 1 to 3) in the absence of BFA. In the presence of BFA, pretreatment with 1-butanol or PA (30 min, before the application of BFA and 1-butanol or PA) completely or largely blocked PIN2 accumulation in BFA compartments (panels 4 and 6). By contrast, in cells pretreated with 2-butanol, extensive PIN2 accumulation in BFA compartments was observed (panel 5). In addition, after pretreatment with BFA for 2 h and BFA washout with PA (panel 9) or 2-butanol (panel 8) for 2 h, the polar localization of PIN2 was almost completely restored, whereas there was still accumulated PIN2-EGFP in cells after washout of BFA with 1-butanol for 2 h (panel 7). Roots of 7-d-old Arabidopsis seedlings were used for all treatments (n > 15). Bars = 5 μm.