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First published online January 26, 2007; 10.1105/tpc.106.041426 The Plant Cell 19:281-295 (2007) © 2007 American Society of Plant Biologists
Arabidopsis PLD
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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2, which is expressed in various tissues and enhanced by auxin. A PLD2-defective mutant, pld2, and transgenic plants deficient in PLD2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29°C, whereas transgenic seedlings overexpressing PLD2 showed opposite phenotypes, suggesting that PLD2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the ß-glucuronidase (GUS) expression in crosses between pld2 and lines containing DR5-GUS indicated that PLD2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLD2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLD2 overexpression. Analyses of crosses between pld2 and lines containing PIN-FORMED2 (PIN2)–enhanced green fluorescent protein showed that PLD2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLD2 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). Both PLD and PA play key roles in plant growth and development, stress responses, and hormone effects (Wang, 2005). PLD-deficient plants or plants treated with the PLD-specific inhibitor 1-butanol showed arrested growth and development, inhibited seed germination and emergence of radicle and cotyledons, and disorganized networks of microtubules and actin (Dhonukshe et al., 2003; Gardiner et al., 2003; Motes et al., 2005; Hirase et al., 2006). PLD1-deficient plants specifically showed altered patterns of root hair formation, whereas PLD1 overexpression resulted in branched and swollen root hairs (Ohashi et al., 2003). PA serves as a second messenger and regulates multiple developmental processes (seedling development, root hair growth and pattern formation, pollen tube growth, leaf senescence, and fruit ripening) and various stress responses (high salinity and dehydration, pathogen wounding, cold, and freezing) (Munnik, 2001; Wang, 2005; Testerink and Munnik, 2005).
In Arabidopsis thaliana, 12 PLD isoforms have been identified that fall into two subfamilies, C2-PLDs and PXPH-PLDs, based on biochemical characteristics and domain structures (Qin and Wang, 2002). Ten members (
, ß, 
, 
, and 
types) belong to the C2-PLD subfamily and contain a conserved Ca2+-dependent phospholipid binding C2 (for protein kinase C–conserved 2) domain at the N terminus and exhibit Ca2+-dependent enzymatic activity (Qin and Wang, 2002; Wang, 2005). The PXPH-PLD members, PLD1 and PLD2, contain PX (for Phox homology) and PH (for Pleckstrin homology) domains at the N terminus and have Ca2+-independent enzymatic activity similar to that of PLDs of yeast and mammalian cells (Qin and Wang, 2002).
Mammalian PLDs and PA are critical in vesicle trafficking, such as vesicle budding from the Golgi apparatus, vesicle transport, exocytosis, endocytosis, and vesicle fusion (Liscovitch et al., 2000; Freyberg et al., 2003; Jenkins and Frohman, 2005). Inhibition of PA biosynthesis by treatment with 1-butanol resulted in inhibited vesicle secretion and complete fragmentation of Golgi membranes in vitro (Siddhanta et al., 2000; Sweeney et al., 2002). PA also regulates vesicle secretion in plants (Monteiro et al., 2005). During pollen tube tip growth, 1-butanol treatment seriously decreased PA amount and the accumulation of PA-mediated secretory vesicles in the apex, resulting in a loss of apical polarity in the pollen tube and inhibited tip growth (Monteiro et al., 2005).
PLD and PA are involved in signaling pathways of plant hormones such as abscisic acid, gibberellic acid, ethylene, and cytokinin. PLD1 and PA participate in abscisic acid–regulated processes through tethering of ABI1 (for ABA-INSENSITIVE1, a negative regulator in the abscisic acid signaling pathway) to the plasma membrane, resulting in reduced ABI1 translocation to the nucleus and inhibited ABI1 function (Zhang et al., 2004). In barley (Hordeum vulgare) aleuronic cells, PLD and PA mediate abscisic acid inhibition of gibberellic acid–promoted -amylase production (Ritchie and Gilroy, 1998). Ethylene stimulates PLD1 expression (Fan et al., 1997), and CTR1, a negative regulator in the ethylene response, is a potential target of PA (Munnik, 2001). 1-Butanol partially blocks cytokinin-induced ARR5–ß-glucuronidase (GUS) expression, indicating PLD involvement in the cytokinin signaling pathway (Romanov et al., 2002). PLD may also play a role in wound-induced jasmonic acid accumulation and jasmonic acid–regulated gene expression (Wang et al., 2000). However, there are still no reports of the participation of PLD in the responses of auxin and brassinosteroids.
Auxin is involved in multiple plant developmental processes, such as the establishment of root and shoot architecture, organ pattern formation, vascular development, gravitropic responses, and cell division/elongation (Cleland, 2004; Woodward and Bartel, 2005). Recent studies showed that auxin regulates the transcription of target genes such as ARF by promoting the degradation of AUXIN/INDOLE ACETIC ACID (Aux/IAA) transcriptional repressors through the auxin receptor TIR1 (Dharmasiri et al., 2005; Kepinski and Leyser, 2005) and ubiquitin-mediated proteolysis (Gray et al., 2001; Tiwari et al., 2001). Mutation or deficiency of auxin signaling proteins such as TIR1, AXR1, and AXR2/IAA7 results in the alteration of auxin-regulated growth processes, including hypocotyl elongation and lateral root formation (Lincoln et al., 1990; Ruegger et al., 1998; Nagpal et al., 2000).
Auxin can be polarly transported from synthesis sites to action sites in a strict directionality through influx and efflux carrier proteins. Influx of auxin is mediated by the influx carrier AUX1, a presumptive auxin permease exhibiting a basal plasma membrane localization (Bennett et al., 1996; Swarup et al., 2001). Efflux of auxin is mediated by several efflux carriers, of which PIN1 is essential for IAA acropetal transport in root tissues (Galweiler et al., 1998) and PIN-FORMED2 (PIN2) is involved in auxin redistribution in root gravitropism (Muller et al., 1998; Shin et al., 2005; Abas et al., 2006). Polar auxin transport is regulated by many factors, including phytotropins (1-N-naphthylphthalamic acid [Katekar and Geissler, 1980]), phosphorylation and dephosphorylation (PID1 [Benjamins et al., 2001] and RCN1 [Rashotte et al., 2001]), and plant hormones (auxin [Paciorek et al., 2005] and brassinosteroids [Li et al., 2005]). Additionally, polar auxin transport depends on actin and vesicle trafficking (Geldner et al., 2001) and is reduced by cytochalasin and the vesicle trafficking inhibitor brefeldin A (BFA) (Cande et al., 1973; Butler et al., 1998; Petrasek et al., 2003). BFA treatment blocks the cycling of PIN1 and PIN2 (Geldner et al., 2001, 2003; Grebe et al., 2003; Shin et al., 2005) between the plasma membrane and the proposed internal pools as well as their accumulation within BFA-induced compartments. Treatment with cytochalasin D, a drug that depolymerizes actin filaments, results in blocked PIN1 accumulation in response to BFA treatment and restoration of PIN1 localization after BFA washout (Geldner et al., 2001).
The mechanism of PIN1 cycling is very similar to the cycling of Glut4 (for glucose transporter 4) in mammalian cells, and both depend on vesicle trafficking and actin (Lachaal et al., 1994; Geldner et al., 2001; Muday and Murphy, 2002). Human PLD1 partially colocalizes with Glut4 and is activated by insulin signaling and trafficking from intracellular membrane storage sites to exocytic sites in Glut4-containing vesicles. Additionally, PA is critical for the process of vesicle fusion to the plasma membrane (Huang et al., 2005). In addition, the inhibition of PA production resulted in severely disorganized actin in Arabidopsis seedlings (Motes et al., 2005), probably because PA binds to ARABIDOPSIS THALIANA CAPPING PROTEIN (ATCP) and inhibits its activity, resulting in enhanced actin polymerization (Huang et al., 2006). Both vesicle trafficking and actin dynamics are important for PIN cycling (Geldner et al., 2001, 2003; Grebe et al., 2003; Shin et al., 2005; Jaillais et al., 2006); however, no plant PLD has been implicated in this process to date.
We show here that PLD2 and PA stimulate auxin responses, root gravitropism, and the expression of early auxin-responsive genes, whereas PLD2 dysfunction results in the opposite responses. Deficiency of PLD2 and 1-butanol treatment reduce auxin accumulation and vesicle trafficking, which could be rescued by exogenously supplied PA. In addition, enhanced PLD2 and PA increase vesicle trafficking and repress BFA compartment formation, stimulating PIN protein cycling and auxin transport. Our data suggest that PLD2 and PA modulate auxin responses through the regulation of vesicle trafficking and auxin transport.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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2, a Putative PXPH-PLD2 (accession number AM182458) and encoding a 1046–amino acid peptide (
118.8 kD), was isolated by PCR-based screening of a cDNA library constructed from Arabidopsis hypocotyl tissues. PLD2 is a single-copy gene, and its deduced protein product PLD2 is 74.4% identical to PLD1, but with little similarity to other Arabidopsis PLD members, including PLD1 (42.4%), PLDß1 (39%), PLD1 (37.5%), and PLD (41.05%).
Structural organization analysis identified several highly conserved domains in PLD2, including two conserved HKD domains (positions 472 to 499 and 847 to 874, with conserved sequences [HxKxxxxD] present in and necessary for all known eukaryotic PLDs) and PX and PH domains (positions 45 to 209 and 216 to 343, respectively) (Figure 1A
), suggesting that PLD2 is a PXPH-PLD. In addition, a PIP2 binding domain, a G-interacting DRY motif, and a conserved IYIENQFF motif were also detected (Figure 1A).
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2 Is Expressed in Various Tissues and Enhanced by IAA2. RT-PCR analysis revealed the expression of PLD2 in seedlings, roots, leaves, stems, and flowers (Figure 1B, top panel). Analysis of GUS activities in several transgenic lines harboring the PLD2 promoter–GUS cassette (pBI101-P) revealed that PLD2 was expressed mainly in seedling cotyledons and rosette leaves (Figure 1B, panels 1 to 4), distal elongation zones of primary and lateral roots (Figure 1B, panels 1 to 3 and 5), vascular and guard cells (Figure 1B, panels 4 and 6), and seeds (Figure 1B, panel 9). PLD2 was highly expressed in anthers, especially pollen grains at late developmental stages (Figure 1B, panels 7 and 8). PLD2 was expressed in embryos (Figure 1B, panels 10 to 12), especially at early developmental stages. These expression profiles are consistent with the results of digital RNA gel blot analysis (https://www.genevestigator.ethz.ch/; Zimmermann, 2004).
RT-PCR analysis further revealed that PLD2 expression was enhanced by auxin. Treatment with 100 µM IAA for 30 min clearly increased the transcript levels of PLD2, with the peak increase after 3 h of IAA treatment (Figure 1C, left panel). Further investigations using transgenic lines expressing the PLD2 promoter–GUS cassette confirmed that auxin treatment enhanced PLD2 expression, which can be detected, for example, at elongation regions of primary and lateral roots (Figure 1C, right panel), suggesting that PLD2 may be involved in auxin signaling and/or action.
The PLD2 Knockout Mutant, pld2, Is Less Sensitive to Exogenous Auxin
A putative PLD2 mutant (SALK_119084) was identified (from the T-DNA insertion library database of the Salk Institute: http://signal.salk.edu/cgi-bin/tdnaexpress) (Alonso et al., 2003), and T-DNA insertion was confirmed at the third intron (14 bp to the left of the fourth exon) (Figure 2A
, top panel) by PCR analysis using primers located in the T-DNA and flanking the genomic DNA. The homozygous mutant line was further identified and confirmed as a knockout mutant by RT-PCR analysis of the defective PLD2 expression (Figure 2A, bottom panel).
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2 was primarily and highly expressed in roots, we focused our analysis on the potential effects of PLD2 on primary root growth. Phenotypic analysis showed that pld2 roots were shorter than those of wild-type seedlings (see Supplemental Figure 1A online) and, importantly, were less sensitive to exogenous auxin. Compared with wild-type seedlings, whose roots were evidently inhibited by exogenous auxin, pld2 roots exhibited much less inhibition (Figure 2B; see Supplemental Figure 1B online). At an IAA concentration of 10 nM, the root length of wild-type seedlings was 79.3% of that of untreated seedlings, whereas pld2 roots showed almost no inhibition. When auxin was supplemented at a concentration of 100 nM, wild-type roots were severely inhibited to 61.3% of the length of untreated seedlings. By contrast, pld2 roots displayed much less inhibition, with root length being 84.8% of that of untreated controls. However, the resistance of pld2 roots to external auxin was not as strong as that of the auxin-insensitive mutant tir1-1 (Ruegger et al., 1998) (Figure 2B), indicating that pld2 has reduced sensitivity, rather than being insensitive, to external auxin.
Previous studies have indicated that mutants with altered auxin signaling (axr2/iaa7 [Nagpal et al., 2000] and axr5-1/iaa1 [Yang et al., 2004]) or transport (aux1 [Bennett et al., 1996], pin2 [Muller et al., 1998], and rcn1 [Rashotte et al., 2001]) often showed altered gravity responses. Measurement of root curvature in gravity responses showed that pld2 was less sensitive to gravity (pld2 bent more slowly than the wild type; Figure 2C). In addition, stimulation of hypocotyl elongation under high temperature (29°C), an important auxin-dependent response (Gray et al., 1998), was suppressed in pld2, although not as strongly as in axr1-3 (Lincoln et al., 1999) (Figure 2D). These observations add evidence for repressed auxin responses of pld2.
Altered Expression of PLD2 Results in Modified Sensitivities to Auxin
Transgenic approaches were further used to study the physiological functions of PLD2. The coding region of PLD2 was subcloned into the modified pCAMBIA1301 binary vector (Liu et al., 2003) under the control of the cauliflower mosaic virus 35S promoter in the sense and antisense orientations, and the resulting cassettes were individually transferred into wild-type Arabidopsis through genetic transformation. In total, 19 sense and 13 antisense independent T1 transgenic lines were obtained, and transcriptional analysis revealed the enhanced (Figure 3A
, left panel) or suppressed (Figure 3A, right panel) expression of PLD2. Homozygous lines containing single-copy T-DNA insertions were obtained and used for subsequent analysis. As PLD1 shares high similarity with PLD2 in nucleotide sequence (64.67%), PLD1 transcript levels were evaluated, and the results showed that transcript levels of PLD1 in antisense plants did not differ substantially from those of controls (Figure 3A, right panel), indicating the specific antisense suppression of PLD2.
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2, the primary roots of antisense seedlings were shorter than those of controls, whereas those of sense plants were significantly longer (see Supplemental Figure 2A online). Statistical analysis showed that the inhibitory effects of auxin on root growth were enhanced in PLD2-overexpressing plants but suppressed in antisense plants (Figure 3B; see Supplemental Figure 2 online). At 10 nM IAA, the primary roots of PLD2-deficient plants were similar to those of untreated controls, whereas those of control and PLD2-overexpressing plants were 82.1%, 68.9% (pO-PLD2-LO11), and 55.6% (pO-PLD2-LO19) as long as those of their respective untreated controls. At 100 nM IAA, primary roots of PLD2-deficient, control, and PLD2-overexpressing plants were 80.9% (pA-PLD2-LA3), 78.1% (pA-PLD2-LA11), 60.6%, 48.7% (pO-PLD2-LO11), and 33.8% (pO-PLD2-LO19) as long as those of their respective untreated controls. At an even higher auxin concentration (1 µM IAA), primary roots of control and PLD2-overexpressing plants were 15% as long as those of their respective untreated controls, whereas those of PLD2-deficient plants were 31.7% (pA-PLD2-LA3) and 44.3% (pA-PLD2-LA11) as long as those of the untreated controls (Figure 3B). These results are consistent with the results for pld2 and indicate that deficiency or overexpression of PLD2 results in less sensitive or hypersensitive responses, respectively, to auxin. In addition, compared with controls, PLD2-overexpressing and -deficient transgenic plants exhibited enhanced and reduced root gravitropism, respectively (Figure 3C).
PA Enhances the Root Gravity Response
Previous studies have indicated reduced PA levels in pld2 (Cruz-Ramirez et al., 2006; Li et al., 2006); thus, we examined the effects of PA on gravitropism. As shown in Figure 4A
, PA (10 µM) stimulated gravitropic responses, whereas inhibiting PA production by 1-butanol (0.2 or 0.4%, v/v) resulted in severely decreased root gravitropism. Two analogs of 1-butanol, 2-butanol (0.4%, v/v) and 3-butanol (0.4%, v/v), displayed no effects on root gravitropism (Figure 4B), indicating that PA acts positively on root gravitropism, in agreement with the increased gravity response shown by PLD2-overexpressing plants.
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2 Enhances Auxin Distribution and Responses2 expression or supplemented PA. Arabidopsis seedlings containing the DR5-GUS cassette (the GUS-coding sequence under transcriptional control of a synthetic auxin-inducible promoter), which has been used widely to detect auxin accumulation and distribution (Ulmasov et al., 1997), was used for this series of experiments. Through genetic crossing, the DR5-GUS cassette was transferred into pld2 and PLD2-overexpressing lines. In control seedlings, GUS signals were detected mainly in root and cotyledon tips, but they were suppressed in the pld2 background, especially in cotyledons. On the other hand, GUS signals were enhanced in the PLD2 overexpression background, with the staining occurring at cotyledon edges and in the root elongation region (Figure 5A
).
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PLD2 Activates the Expression of Auxin-Responsive Genes
IAA5, IAA19, and GH3-3, which belong to the Aux/IAA and GH3 families, were selected to examine whether altered PLD2 expression would affect the transcription of early auxin-responsive genes. Quantitative real-time RT-PCR analysis showed that transcript levels of IAA5, IAA19, and GH3-3 were rapidly upregulated after auxin treatment in wild-type plants, consistent with previous reports (Tian et al., 2002; Nakamura et al., 2003). By contrast, in pld2 or PLD2-deficient antisense plants, the upregulation of these three genes in response to external IAA was suppressed. In PLD2-overexpressing plants, the auxin-induced transcription increases of IAA5, IAA19, and GH3-3 were generally much greater compared with those in wild-type plants (Figure 6
), providing further support for the involvement of PLD2 in controlling auxin responses.
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2 and PA Positively Regulate Vesicle Trafficking2 in vesicle trafficking was investigated using FM4-64, a water-soluble and membrane-selective fluorescent dye that has frequently been used as a membrane marker to monitor endocytosis in mammalian, fungal, and plant cells (Bolte et al., 2004). As depicted in Figure 7
, when wild-type seedling roots were submerged in FM4-64 solution, the dye was soon internalized (Figure 7A, panels 1 and 6). But in pld2 and PLD2-deficient plants, the internalization of FM4-64 into root cells was severely reduced (Figure 7A, panels 2, 5 and 7, 10, respectively). On the contrary, FM4-64 internalization into root cells of PLD2-overexpressing plants was enhanced, with many more fluorescently labeled vesicles (Figure 7A, panels 3, 4, 8, and 9).
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2 (see Supplemental Figure 4 online, panels 2 and 3). These findings suggest that PLD2 and PA positively regulate vesicle trafficking in Arabidopsis root cells.
PLD2 Suppresses BFA Compartment Formation and BFA Inhibition on PIN1 and PIN2 Cycling
BFA is a vesicle transport inhibitor that specifically blocks cell exocytosis but allows endocytosis, resulting in the internalization and accumulation of recycling plasma membrane proteins in BFA compartments (Nebenfuhr et al., 2002). Many plasma membrane proteins, including PIN1, plasma membrane H+-ATPase (Geldner et al., 2001, 2003), and PIN2 (Geldner et al., 2003; Grebe et al., 2003) have been found in BFA compartments. Based on results showing that altered PLD2 expression or PA levels modified vesicle trafficking, we examined BFA compartment formation in wild-type plants and plants with altered PLD2 levels. After BFA treatment (50 µM) for 2 h, BFA compartments were observed in almost every cell of wild-type seedling roots (Figure 8A
, panel 1) and in nearly all root cells of pld2 (Figure 8A, panel 4) and PLD2-deficient (Figure 8A, panels 5 and 6) seedlings, although the size was much smaller. On the other hand, BFA compartments were much fewer in root cells (30 to 40%) of PLD2-overexpressing seedlings (Figure 8A, panels 2 and 3).
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, 2003). Indeed, compared with wild-type seedlings, statistical analysis showed that PLD2-overexpressing seedlings were less sensitive to BFA inhibition of elongation of hypocotyls (see Supplemental Figure 5 online) and primary roots, lateral root formation, and root gravity response (Figures 8B and 8C). However, pld2 and wild-type seedlings did not differ significantly in their sensitivity to BFA inhibition.
Altered auxin sensitivity and auxin-regulated hypocotyl elongation, root gravity, and DR5-GUS expression in PLD2 mutant or overexpression lines suggested altered auxin signaling or auxin transport. It was shown that cycling of the auxin efflux carriers PIN1 and PIN2 is vesicle-dependent (Geldner et al., 2001, 2003; Grebe et al., 2003; Jaillais et al., 2006) and that BFA treatment reduced auxin transport (Petrasek et al., 2003). As PLD2 and PA positively regulate vesicle trafficking and suppress BFA inhibition, the effects of PLD2 deficiency or overexpression on the cycling of PIN2 between plasma membrane and endosomal compartments were then examined. To achieve this, PIN2–enhanced green fluorescent protein (EGFP) (Xu and Scheres, 2005) expression cassettes were individually transferred into a pld2 background and into PLD2-overexpressing lines through genetic crossing. Figure 9A
shows that PLD2 deficiency or overexpression had no effect on the polar localization of PIN2 (Figure 9A, panels 2 to 4). When treated with BFA for 2 h, the cycling of PIN2-EGFP was blocked, with the appearance of two or three large BFA compartments (Figure 9A, panel 5). In pld2 or PLD2-overexpressing lines, the BFA-induced cytoplasmic aggregation of PIN2-EGFP was generally suppressed (Figure 9A, panels 6 to 8). After washout for 2 h, BFA compartments disappeared and normal polar localization of PIN2-EGFP was detected in the wild-type background (Figure 9A, panel 9) and in PLD2-overexpressing seedlings (Figure 9A, panels 11 and 12). On the other hand, accumulated PIN2-EGFP in pld2 could not be resumed effectively (Figure 9A, panel 10), suggesting a positive effect of PLD2 on PIN2 cycling.
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2, accumulation of PIN2-EGFP (albeit smaller and fewer in number) could still be observed in PLD2-overexpressing plants under BFA treatment, suggesting partial repression of the BFA inhibitory effect. Indeed, PIN2 cycling was stimulated in PLD2-overexpressing plants (Figure 9A, panel 12), consistent with previous results indicating that overexpression of PLD2 partially decreased BFA effects on seedling growth (Figure 8B; see Supplemental Figure 5 online). These observations were further confirmed by investigating PIN2-EGFP localization with PA or 1-butanol treatment. As Figure 9B shows, 1-butanol and PA had no effect on the polar localization of PIN2-EGFP (Figure 9B, panels 1 to 3); however, after pretreatment with 1-butanol or PA (30 min) and application of BFA and 1-butanol or PA (2 h), the BFA inhibition of PIN2-EGFP cycling was completely or largely blocked (Figure 9B, panels 4 and 6). By contrast, pretreatment with 2-butanol had no obvious influence (Figure 9B, panel 5). In addition, after cells were treated with BFA for 2 h, BFA washout with 1-butanol (2 h) resulted in differential accumulation of PIN2-EGFP compared with that with 2-butanol or PA. The intracellular compartments disappeared, and polar localization of PIN2-EGFP was effectively restored in cells with 2-butanol (Figure 9B, panel 8) or PA (Figure 9B, panel 9), whereas with 1-butanol, intracellular compartments did not disappear completely and the polar localization of PIN2-EGFP was not effectively recovered (Figure 9B, panel 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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1 and PLD2 are unique in possessing the PX and PH domains. Although considerable progress has been made in understanding the genetic function and biochemical properties of PLD1, only limited knowledge has been obtained on PLD2. Previous investigations revealed the important role of PLD2 in phosphate starvation responses of Arabidopsis (Cruz-Ramirez et al., 2006; Li et al., 2006). In this work, we show that PLD2 is involved in vesicle trafficking in higher plants, and our studies reveal the role and mechanisms of PLD2 in regulating auxin responses, providing hints of crosstalk between the phosphatidylinositol signaling and auxin signaling pathways.
PLD2 and PA Positively Regulate Auxin Responses
In agreement with previous investigations, we found that PLD2 is expressed in multiple tissues. We also found that PLD2 expression is clearly and significantly increased in response to auxin treatment. Two categories of evidence support the finding that PLD2 positively regulates auxin responses in Arabidopsis, and this regulation may be achieved through PA, the product of PLD2. Genetic analysis revealed that PLD2-deficient mutants (knockout and antisense lines) exhibited suppressed auxin responses, whereas PLD2-overexpressing plants showed the opposite phenotype. These results were confirmed by a variety of approaches (i.e., sensitivity to inhibition of root growth by external auxin, alteration in gravity responses, stimulation of hypocotyl elongation at 29°C, size and distribution of the DR5-GUS expression region, and transcriptional pattern of early auxin-responsive genes). In addition, consistent with the positive effects of enhanced PLD2 on auxin responses, treatment of wild-type seedlings with PA stimulated gravitropism and expanded the expression regions of DR5-GUS. The data obtained with PA are relevant to the function of PLD2, because the stimulating effects on auxin responsiveness observed in PA application were reversed in seedlings treated with 1-butanol and in PLD2-defective mutants (knockout and antisense plants). This finding provides strong evidence that PLD2 regulates auxin responses through its product PA. However, there is still no evidence that PLD2 and PA are involved directly in auxin signaling.
At present, we cannot exclude the possibility that, in vivo, PA produced by other PLD or PLC members does not participate in auxin responses. Inhibiting PA by 1-butanol treatment repressed auxin-induced lateral root formation (see Supplemental Figure 3B online) and root gravitropic responses (Figure 4B) more strongly than those of pld2, indicating that other PLD members or PLC may be involved in auxin responses. Further studies to examine whether other PLD or PLC members contribute to auxin responses and whether there are functional interactions between PLD2 and other PLD or PLC members in regulating auxin responses would provide much information.
PLD2 and PA Enhance Vesicle Trafficking
Many previous studies have documented that PLDs and PA are involved in vesicle trafficking in both mammalian and plant cells (Liscovitch et al., 2000; Freyberg et al., 2003; Jenkins and Frohman, 2005; Monteiro et al., 2005). In addition, PLD and PA are important for vesicle fusion and aggregation (Jones and Clague, 1997; Ichikawa and Walde, 2004; Huang et al., 2005; Jenkins and Frohman, 2005). In the process of sporulation in yeast, loss of Spo14P, the single phosphatidylcholine-specific PLD, blocks vesicle fusion, resulting in the accumulation of prospore membrane precursor vesicles (Nakanishi et al., 2006). Treatment with 1-butanol or decreased PLD1 activity inhibited early endosome fusion or vesicle fusion to the plasma membrane, which was stimulated by exogenous PLD or lysophosphatidylcholine (Jones and Clague, 1997; Huang et al., 2005). Suppressed vesicle trafficking under PLD2 deficiency might reduce vesicle fusion and aggregation, leading to the formation of small BFA compartments.
We show that PLD2, or PA generated by it, stimulates vesicle trafficking in Arabidopsis cells. Genetic investigations using PLD2-deficient or -overexpressing plants demonstrate unequivocally that PLD2 is beneficial for internalization of the membrane marker FM4-64 through vesicle trafficking. Significantly, exogenously applied PA mimics the effect of PLD2 overexpression on FM4-64 internalization, whereas 1-butanol treatment diminishes FM4-64 internalization in a way similar to PLD2 deficiency. These findings are consistent with previous results showing that decreased PA in the pld2 mutant (Cruz-Ramirez et al., 2006) reduced the accumulation of secretory vesicles in the pollen tube apex. As a widely accepted tracer for endocytic trafficking (Bolte et al., 2004), the reduced internalization of FM4-64 in pld2 and wild-type seedlings treated with 1-butanol suggests that PLD2 and PA at least affected endocytosis (Figure 7).
PLD2 overexpression significantly counteracts the inhibitory effect of BFA on vesicle trafficking, further confirming PLD2 function in stimulating vesicle trafficking. The alleviation of BFA inhibition on vesicle trafficking by PLD2 overexpression correlates well with the significantly improved performance of PLD2-overexpressing plants in terms of hypocotyl elongation, primary root length, lateral root development, and gravity response on medium containing BFA.
Stimulation of PIN2 Cycling Underlies the Positive Roles of PLD2 and PA in Auxin Transport and Enhanced Auxin Responses
Polar auxin transport, which is essential for the execution of auxin responses, is mediated by plasma membrane–localized auxin influx and efflux carrier proteins, and PINs were the best characterized proteins controlling auxin efflux (Muday and Delong, 2001; Muday and Murphy, 2002). The cycling of PIN proteins (PIN1 and PIN2) between membrane and endosomal compartments is vital for their transport function and is dependent on vesicle trafficking (Geldner et al., 2001, 2003; Grebe et al., 2003; Jaillais et al., 2006). PIN1 cycling and auxin transport require endosomal GNOM activity (Geldner et al., 2003), and recent studies have shown that Arabidopsis SORTING NEXIN1, which localizes to an endosomal compartment distinct from GNOM-containing endosomes, was involved in PIN2 endocytic sorting (Jaillais et al., 2006).
Through complementary genetic and cell biological analyses, we deduced that a positive role of PLD2 (and PA) in auxin responses is at least partly linked with its beneficial effect on the rapid cycling of PIN2 in cells. Compared with wild-type and PLD2-deficient cells, the formation of BFA compartments containing PIN2 was substantially suppressed in PLD2-overexpressing cells. More importantly, after BFA removal, the resumption of PIN2 polar localization was more rapid and complete under enhanced PLD2 expression. The effect of exogenous PA was similar to that of PLD2 overexpression in facilitating PIN2 cycling. Together, these results indicate that PLD2 (through its product PA) is required for the normal cycling of PIN2-containing vesicles and regulates auxin transport, which may account for the role of PLD2 in positively regulating auxin responses.
It is noteworthy that, although both PA and 1-butanol seem to alleviate the effects of BFA on PIN protein cycling, the relevant mechanisms are different. PA represses BFA inhibition of exocytosis and restores BFA compartments (accumulated PIN proteins) through accelerated vesicle trafficking, whereas the reduced BFA compartments under 1-butanol pretreatment are attributable to inhibited endocytosis. The restoration of BFA compartments after BFA washout further confirms the different mechanisms.
Our analyses showed that PLD2 is involved in auxin responses and the regulation of auxin levels and distribution, suggesting that altered responses may be attributable to altered auxin transport and thus changed auxin distribution and levels. This is confirmed by the observation that PIN protein cycling was altered under modulated expression of PLD2. Altered PIN protein cycling and auxin transport result in altered auxin distribution and levels, consequent to modulated auxin responses, as well as the expression of early auxin-responsive genes. However, there is no evidence that PLD2 and PA are involved directly in auxin signaling. These results suggest that PLD2 may indirectly control auxin signaling by altering auxin transport and are consistent with the fact that pld2 showed decreased sensitivity to auxin but not the striking phenotype of the auxin signaling–defective mutants tir1-1 and axr1-3.
Mechanistically, PLD and PA may stimulate PIN protein cycling through actin regulation. Inhibited PLD activity resulted in severely disorganized actin structures in Arabidopsis seedlings (Motes et al., 2005), implying an essential role of PLD and PA for actin. In addition, PLD can bind to actin (Kusner et al., 2002), and PA can stimulate actin polymerization by inhibiting ATCP activity (Huang et al., 2006). PIN1 cycling is actin-dependent, and treatment with cytochalasin, a drug that depolymerizes actin filaments, reduced auxin transport (Cande et al., 1973; Butler et al., 1998), blocked PIN1 accumulation in response to BFA treatment, and restored PIN1 localization after BFA was washed out (Geldner et al., 2001). Cytochalasin D and 1-butanol have similar inhibitory effects on PIN cycling. Each of them has only a slight effect on the polar localization of PIN, but both inhibit BFA-induced PIN accumulation and PIN trafficking from BFA compartments to the plasma membrane.
PLD and PA may be involved in auxin transport through some other mechanism, such as regulating the activity of RCN1 or PID1. RCN1, a protein phosphatase 2A regulatory subunit involved in auxin transport (Rashotte et al., 2001), is a potential target protein of PA (Testerink et al., 2004). PID1, a Ser/Thr protein kinase, acts as a regulator of polar auxin transport (Bennett et al., 1995), is activated by Arabidopsis PDK1 (3-phosphoinositide–dependent protein kinase 1 [Zegzouti et al., 2006]), and its activity is stimulated directly by PA (Anthony et al., 2004). rcn1 showed increased auxin transport and decreased root gravitropism (Rashotte et al., 2001), and pid1 has similar phenotypes to those of pin1 (Bennett et al., 1995). Overexpression of PID1 results in phenotypes similar to those of auxin-insensitive mutants (Christensen et al., 2000) and induces a basal-to-apical shift of PIN1 localization (Friml et al., 2004). These results suggest that PLD and PA may be involved in auxin transport regulation through modulation of the activity of RCN1 or PID1.
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Bacterial and Plant Materials and Growth Conditions
Escherichia coli strain XL1-Blue MRF was used for DNA amplification and subcloning. Agrobacterium tumefaciens strain GV3101 was used for plant transformation. Arabidopsis thaliana ecotype Columbia was used for transformation by the floral dip method (Clough and Bent, 1998). Seeds were germinated and seedlings were grown in a phytotron set at 22°C with a 16-h light/8-h dark cycle. Ten to 14 d after germination, seedlings were hand-transferred to soil and grown in a phytotron under the same conditions. For hormone treatments, 7-d-old seedlings were treated with 100 µM IAA for various times (0, 0.5, 1, 3, 6, and 12 h).
Isolation of PLD2 cDNA
An Arabidopsis cDNA clone encoding a putative PLD2 was identified through analysis of the Arabidopsis genomic sequence. Specific primers PLD2-1 (5'-GATCGTTATTCCGCTAC-3') and PLD2-2 (5'-AAGGTTCCCTCTT
2 Regulates Vesicle Trafficking and Is Required for Auxin Response
