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Plant Cell. Jun 2011; 23(6): 2184–2195.
Published online Jun 3, 2011. doi:  10.1105/tpc.111.086355
PMCID: PMC3160035

Gibberellin Regulates PIN-FORMED Abundance and Is Required for Auxin Transport–Dependent Growth and Development in Arabidopsis thaliana[C][W]

Abstract

Plants integrate different regulatory signals to control their growth and development. Although a number of physiological observations suggest that there is crosstalk between the phytohormone gibberellin (GA) and auxin, as well as with auxin transport, the molecular basis for this hormonal crosstalk remains largely unexplained. Here, we show that auxin transport is reduced in the inflorescences of Arabidopsis thaliana mutants deficient in GA biosynthesis and signaling. We further show that this reduced auxin transport correlates with a reduction in the abundance of PIN-FORMED (PIN) auxin efflux facilitators in GA-deficient plants and that PIN protein levels recover to wild-type levels following GA treatment. We also demonstrate that the regulation of PIN protein levels cannot be explained by a transcriptional regulation of the PIN genes but that GA deficiency promotes, at least in the case of PIN2, the targeting of PIN proteins for vacuolar degradation. In genetic studies, we reveal that the reduced auxin transport of GA mutants correlates with an impairment in two PIN-dependent growth processes, namely, cotyledon differentiation and root gravitropic responses. Our study thus presents evidence for a role of GA in these growth responses and for a GA-dependent modulation of PIN turnover that may be causative for these differential growth responses.

INTRODUCTION

Throughout their development, plants have to integrate a multitude of internal and external signals to adapt their growth to the environment and to guarantee reproductive success. Elucidating the molecular mechanisms of such integration is a central topic in plant biology. In this article, we examine how the plant hormone gibberellin (GA) regulates processes that require the directional transport of the plant hormone auxin and how GA thereby modulates auxin-dependent growth during Arabidopsis thaliana development.

The phytohormone auxin (indole-3-acetic acid [IAA]) regulates plant morphogenesis during all stages of development, integrating environmental light and gravity signals to allow directional growth during phototropic and gravitropic responses (Benjamins and Scheres, 2008). At the cellular level, auxin mediates the expression of auxin-responsive genes through the inactivation of AUX/IAA transcriptional repressors that negatively regulate the activity of AUXIN REPONSE FACTOR transcription factors (Chapman and Estelle, 2009). At the organismal level, auxin is transported through the plant body by a system of AUXIN RESISTANT1 (AUX1)/LIKE-AUX1 (LAX) (Bennett et al., 1996; Müller et al., 1998; Marchant et al., 1999; Yang et al., 2006; Bainbridge et al., 2008) and PIN-FORMED (PIN) proteins (Kramer and Bennett, 2006; Petrásek et al., 2006; Teale et al., 2006; Vieten et al., 2007) from the sites of auxin biosynthesis to sites of auxin action. In the context of this directional auxin transport, AUX1/LAX proteins have been shown to function as auxin influx facilitators, while PIN proteins are critical facilitators of auxin efflux that are thought to function together with members of the family of MULTIDRUG RESISTANCE/PHOSPHO-GLYCOPROTEIN proteins (Chen et al., 1998; Luschnig et al., 1998; Geisler and Murphy, 2006; Petrásek et al., 2006; Yang et al., 2006; Bandyopadhyay et al., 2007; Blakeslee et al., 2007). Through directional auxin transport and the resulting formation of local auxin maxima or minima, auxin can influence a diverse array of developmental processes, such as organ differentiation in the shoot, root differentiation in the root meristem, and lateral root formation along the differentiated root, as well as tropic responses, such as phototropism and gravitropism.

PIN auxin efflux facilitators are particularly interesting because they have a polar distribution in the plasma membrane of many cells and because their polar distribution apparently allows the prediction of directional auxin transport within the plant body (Grieneisen et al., 2007; Wabnik et al., 2010). At least five PIN family members, namely, PIN1, PIN2, PIN3, PIN4, and PIN7, participate in the cell-to-cell transport of auxin in Arabidopsis. These PIN proteins have redundant functions, and mutants lacking multiple PIN genes have severe growth and differentiation defects (Blilou et al., 2005; Vieten et al., 2005). Among the five PIN family members, PIN1 and PIN2 are particularly well suited for developmental and genetic studies because their loss-of-function mutants have specific phenotypes that can be attributed to the loss of specific auxin-dependent developmental processes: PIN1 is the major auxin efflux facilitator in the Arabidopsis shoot and loss of PIN1 impairs cotyledon differentiation, phyllotaxy, and shoot differentiation, thereby displaying the name-giving pin-shaped inflorescences of the pin1 mutant (Gälweiler et al., 1998). PIN2 regulates primarily root gravitropism, and roots of pin2 mutants are fully agravitropic (Luschnig et al., 1998; Müller et al., 1998; Utsuno et al., 1998). The latter is the consequence of pin2 mutants being unable to redistribute auxin via epidermis- and lateral root cap-localized PIN2 proteins so that they cannot generate a lateral auxin maximum at the site of root bending (Ottenschläger et al., 2003).

GA is another important plant hormone and it affects a number of central developmental responses, including germination, elongation growth, and flowering time (Ueguchi-Tanaka et al., 2007a; Schwechheimer, 2008). GA binds to GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors and thereby activates E3 ubiquitin ligases, such as Arabidopsis SCFSLEEPY1(SLY1) and SCFSNEEZY(SNE), which promote the ubiquitylation and ultimately proteolytic degradation of DELLA proteins (Ueguchi-Tanaka et al., 2007b; Willige et al., 2007; Shimada et al., 2008). DELLA proteins are repressors of transcription regulators, such as the PHYTOCHROME INTERACTING FACTOR family transcription factors, ALCATRAZ, or the JASMONIC ACID ZIM-domain protein (de Lucas et al., 2008; Feng et al., 2008; Arnaud et al., 2010; Hou et al., 2010). Thus, as far as GA action is understood today, GA regulates a transcription factor cascade through the consecutive action of posttranslational and transcriptional derepression events. However, how GA and GA signaling lead to the execution of individual GA responses at the cellular level is not understood.

A number of physiological observations suggest a crosstalk between GA signaling and auxin transport. For example, it has been shown that impaired auxin transport leads to the stabilization of the DELLA repressor REPRESSOR-OF-ga1-3 (RGA) of the GA pathway in Arabidopsis and that DELLA proteins delay root growth in response to the loss of shoot-derived auxin (Fu and Harberd, 2003). Since it has also been reported that auxin can induce the transcription of various GA biosynthesis genes (Frigerio et al., 2006), it may be inferred from this observation that a reduction in GA biosynthesis is causative for a stabilization of DELLA proteins in cells that have reduced auxin levels due to impairments in auxin transport. In turn, reports from various plant species suggest that GA promotes auxin transport: It was reported already in the 1960s that GA application leads to an increase in auxin transport that correlates with an increase in apical dominance in pea (Pisum sativum) (Jacobs and Case, 1965). More recently, it was reported that Arabidopsis mutants with reduced GA responses have reduced apical dominance (Silverstone et al., 1997), which could be explained physiologically by reduced auxin transport in the primary inflorescence. In line with this hypothesis, a more recent study with poplar (Populus tremula × tremuloides) shows that exogenous GA application stimulates auxin transport (Björklund et al., 2007). A genetic study conducted in Arabidopsis furthermore revealed that the GA pathway, directly or indirectly, represses the transcription of ARR1 at 5 d after germination in the transition zone of the root meristem (Moubayidin et al., 2010). ARR1 is activated by the cytokinin response pathway and represses the expression of the PIN genes by promoting the transcription of the auxin response regulator SHY2. Thereby, GA and DELLA proteins are seemingly implicated in the control of auxin transport and the coordination between cell division and cell differentiation during this stage of root growth and differentiation (Moubayidin et al., 2010). Taken together, the above-cited studies and observations from different plant species strongly suggest a crosstalk between GA and auxin transport. However, how precisely GA interferes at the molecular and cellular level with auxin transport is not understood as yet.

Here, we present physiological, cell biological, and developmental data that establish that GA is required for proper auxin transport in Arabidopsis. We show that the auxin transport impairment in GA mutants can be explained by a decrease in PIN auxin efflux facilitator abundance and reveal that the GA pathway interacts with PIN protein–dependent auxin transport and development during embryogenesis and root gravitropic responses.

RESULTS

Auxin Transport Is Reduced in GA Mutants

As part of a physiological characterization of GA mutants from Arabidopsis, we performed auxin transport measurements using sections from primary inflorescences of GA biosynthesis and signaling mutants: (1) ga1 (SALK_109115; ecotype Columbia [Col]) is a GA REQUIRING1 (GA1) loss-of-function allele that is impaired in an early step of GA biosynthesis due to an insertion in the gene encoding ENT-COPALYL DIPHOSPHATE SYNTHETASE1 (Sun and Kamiya, 1994; Willige et al., 2007). Since this ga1 allele normally does not bolt, the mutant was treated with gibberellic acid (GA3) to suppress its GA deficiency and to induce bolting. GA3 treatment was then omitted for 3 weeks to deplete the mutant of GA before auxin transport was measured from mutant inflorescences. (2) gid1ac (ecotype Col) is a loss-of-function mutant of two of three genes encoding the GID1 GA receptor (Willige et al., 2007). gid1ac is the most severe GID1 receptor mutant available for these studies because the GID1-deficient gid1abc mutant does not produce a stem and its GA response defect cannot be rescued by GA treatments (Willige et al., 2007). (3) gai-1 (ecotype Landsberg erecta) is a dominant gain-of-function allele of the DELLA protein gene GIBBERELLIC ACID INSENSITIVE (GAI). gai-1 constitutively represses GA signaling because it is unable to interact with the GA-bound GID1 due to a mutation in the DELLA domain required for GID1 interaction (Peng et al., 1997; Willige et al., 2007). When we examined each of the above-mentioned GA mutants with regard to auxin transport, we noted with interest that each of the tested mutants is compromised in auxin transport when compared with the respective Col and Landsberg erecta wild types (Figure 1). This suggests that proper GA biosynthesis and signaling are required for proper auxin transport.

Figure 1.
GA Biosynthesis and Signaling Are Required for Proper Auxin Transport.

GA Is Required for PIN Protein Accumulation in Stems and Root Tips

Directional auxin transport requires the activities of AUX1/LAX auxin influx carriers and PIN auxin efflux facilitators. Since the reduction in auxin transport observed in GA mutants may be caused by differences in the abundance of these transport proteins, we next sought to examine PIN and AUX1 protein distribution and abundance in the wild type and in GA-deficient backgrounds. To this end, we looked at the cellular abundance and intracellular distribution of the auxin influx carrier AUX1 and the auxin efflux facilitators PIN1, PIN2, or PIN3 in the roots of transgenic lines expressing fluorescent protein–tagged versions of these proteins in solvent control-(mock-)treated seedlings and following treatments with the GA biosynthesis inhibitor paclobutrazol (PAC) (Rademacher, 2000). Via live-cell imaging, we did not observe any obvious differences in the abundance or distribution of AUX1:yellow fluorescent protein (YFP) following PAC treatment (see Supplemental Figure 1 online). However, we observed a considerable reduction in the abundance of PIN1:green fluorescent protein (GFP), PIN2:GFP, and PIN3:GFP in the meristematic zone of roots after PAC treatment (Figure 2A). We then introgressed the PIN:PIN:GFP transgenes into the ga1 background and could subsequently confirm the GA dependency of PIN protein abundance also in this GA-deficient background (Figure 2A). Neither in PAC-treated wild-type seedlings nor in the ga1 background did we note a change in the polarity of the individual PIN:GFP proteins (see Supplemental Figure 2A online). A quantification of the respective fluorescence intensities revealed a reduction of plasma membrane–localized GFP signals to 18 and 28% in the case of PIN1 (n = 60 stele cells), 6 and 10% for PIN2 (n = 60 epidermal cells), and 37 and 33% of PIN3 (n = 60 stele cells) in the meristematic zone of root tips of PAC-treated seedlings or ga1 mutants, respectively, when compared with the same region of roots from untreated wild-type seedlings (Figure 2B). In each case, the reduction in PIN protein levels could be suppressed by supplying the GA-deficient seedlings with GA3 for 24 h (Figures 2A and 2B) or by growing them on GA3-containing medium (see Supplemental Figure 3 online). Taken together, these findings thus reveal that PIN protein abundance in the meristematic zone of the root is modulated by GA. We further noted that this GA-dependent modulation of PIN levels is restricted to cells of the root meristem and is not observed in the elongation or differentiation zone of the root (see Supplemental Figures 2B and 2C online).

Figure 2.
Cell Biological Evidence for a Reduction of PIN Protein Abundance in GA-Deficient Backgrounds.

We then substantiated the live-cell imaging results on differential PIN protein accumulation in the root using immunoblots of PIN proteins. Because the live-cell imaging data had suggested that PIN protein levels are specifically reduced in the root meristematic zone, we examined the accumulation of PIN1 (and PIN1:GFP) as well as that of PIN2 by immunoblot using protein extracts prepared from entire roots as well as from dissected root tips (1.5 to 2 mm) of wild-type and ga1 mutant seedlings (Figure 3A). In line with the observations made by live-cell imaging that PIN protein levels are dependent on GA only in the root tip, our immunoblot analysis of whole root protein extracts revealed that PIN protein levels are not modulated by the presence or absence of GA (Figure 3B, PIN1; Figure 3D, PIN1:GFP; Figure 3F, PIN2). More importantly, however, we found PIN levels to be reduced when we specifically analyzed protein extracts prepared from root tips (Figure 3C, PIN1; Figure 3E, PIN1:GFP; Figure 3G, PIN2). The quantification of immunoblots from three independent biological replicate samples performed with root tip protein extracts revealed a reduction in PIN protein accumulation to ~70% of the wild-type levels. Specifically, this quantification revealed a reduction for PIN1 to 72.4% ± 11.1% (Figure 3C), for PIN1:GFP to 69.0% ± 11.3% (Figure 3E), and for PIN2 to 68.6% ± 5.4% (Figure 3G) when normalized to the respective loading control and compared with the wild type. That the reductions in PIN protein levels are smaller in the immunoblot analyses (Figure 3) than when observed by live-cell imaging (Figure 2) can be explained by the fact that the meristematic zone where differences in the accumulation of PIN1 and PIN2 are apparent by live-cell imaging is smaller (e.g., 0.5 to 0.6 mm in the case of PIN1:GFP) than what can be manually dissected for the preparation of protein extracts from root tips (1.5 to 2 mm) (Figure 3A). The differential accumulation of PIN protein in the root meristem is therefore partially masked by PIN protein from the region above the meristem. In summary, we conclude that PIN protein levels are reduced in the root meristems of GA-deficient seedlings, inviting the conclusion that auxin transport and particularly auxin efflux is impaired in these seedlings.

Figure 3.
Biochemical Evidence for a Reduction of PIN Protein Levels in GA Mutant Backgrounds.

Since our initial experiments revealed that auxin transport is reduced in inflorescence stem segments of GA biosynthesis and signaling mutants (Figure 1), we also examined whether PIN protein levels are modulated by GA in the shoot. To this end, we examined PIN1 protein accumulation in the inflorescence stems of ga1, gid1ac, and gai-1 mutants. In agreement with the reduced auxin transport observed in these mutants (Figure 1), we found in each case reduced PIN protein accumulation when compared with the wild type (Figures 3H to 3J), inviting the conclusion that reduced PIN1 protein levels may be the cause for the mutants’ reduced auxin transport.

PIN Gene Transcription Cannot Explain Differential PIN Protein Accumulation

The reduction of PIN protein levels in the root meristematic region in the absence of GA may be explained by reduced PIN transcription. We therefore examined PIN gene expression using previously established and characterized PIN:β-glucuronidase (GUS) reporter lines (Vieten et al., 2005). This gene expression analysis showed that the expression of PIN1:GUS in the root tip is increased in the absence of GA and reduced following GA treatment (Figure 4A). Thus, the observed transcriptional regulation of PIN1:GUS does not explain the behavior of the PIN1 protein, which shows the opposite behavior in being reduced in the absence of GA and increased in its presence (Figures 2 and and3).3). The expression of PIN2:GUS was unaltered when the wild type and ga1 were compared, and although PAC treatment of PIN2:GUS led to a reduction of GUS activity in the wild type, this effect was not GA-reversible, indicating that this PAC effect is not related to the effect of PAC on GA biosynthesis (Rademacher, 2000) (Figure 4B). In the case of PIN3:GUS, we did not observe any expression changes in ga1 or following PAC treatment (Figure 4C). PIN4:GUS, which we did not analyze at the protein level, showed the same differential transcriptional regulation as PIN1:GUS, but these differences appeared less pronounced since the activity of this reporter in the wild type is considerably higher than that of PIN1:GUS (Figure 4D). In the case of PIN7:GUS, we noted a moderate and GA-reversible reduction of PIN7:GUS expression in the columella root cap under GA-deficient conditions (Figure 4E). Since we have not analyzed PIN7 distribution in the root, we cannot correlate this observation with the behavior of the corresponding PIN7 protein. Taken together, we conclude from our gene expression analysis of the individual PIN genes that their gene expression behavior does not explain the differential GA-dependent accumulation of the PIN proteins that we observed in the GA biosynthesis and signaling mutants.

Figure 4.
PIN Gene Expression in Root Tips Does Not Explain Differences in PIN Protein Abundance in the Absence of GA Biosynthesis.

GA Deficiency Promotes Vacuolar Degradation of PIN2:GFP

We next turned to examine the cellular behavior of PIN2:GFP, which is already well understood with regard to its intracellular trafficking and its vacuolar turnover (Kleine-Vehn et al., 2008). We examined the vacuolar targeting of PIN2:GFP in the wild type and ga1 mutants using Concanamycin A (ConA), which blocks protein degradation in the vacuole by inhibiting vacuolar H-ATPase activity and vacuole acidification (Páli et al., 2004). In this experiment, we observed more cells with a vacuolar PIN2:GFP signal per seedling in ga1 (experiment 1: 50.3%, n = 230 cells; experiment 2: 56.3%, n = 477 cells) than in the wild type (experiment 1: 41.6%, n = 278 cells; experiment 2: 44.0%, n = 572 cells) (Figures 5A and 5B). The effect of ConA on PIN2:GFP vacuolar targeting was even more pronounced when we examined the effect of the drug using immunoblots of root tips dissected from ConA-treated ga1 and wild-type seedlings (Figure 5C). Whereas PIN2 levels were much lower in the extracts from untreated ga1 seedling root tips (see also Figure 3G), PIN2 accumulated to similar levels in ga1 and the wild type following ConA treatment (Figure 5C). We interpret this finding as demonstrating that similar amounts of PIN2 protein are translated in the wild type and the ga1 mutant but that more PIN2 protein is targeted to the vacuole in ga1, leading ultimately to a reduction of PIN2 abundance in ga1 (Figure 5C). The conclusion that there is a differential GA-dependent targeting of PIN2 to the vacuole is further substantiated by our observation that the basal levels of PIN2 are increased in a GA3-treated ga1 mutant when compared with the untreated (mock) ga1 control (Figure 5D). Importantly, the differential accumulation of PIN2 is also observed in immunoblots when vacuolar protein targeting was inhibited by Wortmannin (WM), a drug that blocks the transport of PIN2 from the prevacuolar compartment to the vacuole (Figure 5E) (Matsuoka et al., 1995). In summary, our data suggest that the decrease of PIN2 levels at the plasma membranes of root tip cells in ga1 is the result of an increased vacuolar targeting of PIN2 in the absence of GA.

Figure 5.
PIN2 Abundance in the Vacuole Is Increased and Hypersensitive to ConA and WM in ga1.

GA Promotes Proper Cotyledon Differentiation together with PIN1

In order to evaluate the contribution of GA signaling to PIN- and auxin transport–regulated development, we subsequently examined the morphology of ga1 mutant seedlings with regard to phenotypes that may be explained by defects in auxin transport (Friml, 2003; Weijers and Jürgens, 2005; Benjamins and Scheres, 2008). pin1 mutants or wild-type embryos treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) have defects in cotyledon morphology, positioning, and number (Gälweiler et al., 1998; Hadfi et al., 1998; Zourelidou et al., 2009). Although these phenotypes appear only with a relatively low penetrance in pin1 mutants (13.3%; n = 97 [of 388 pin1/PIN1]), the occurrence of these phenotypes is commonly explained by defects in auxin transport or signaling (Figures 6A and 6B). We wanted to examine whether GA contributes to auxin transport–dependent processes during embryogenesis but were facing the problem that GA treatment is needed to promote flower development and fertility in the ga1 mutant. To avoid an effect of GA3 treatments on the development of ga1 mutants during embryogenesis, we omitted GA application after flower formation when preparing seeds for the analysis described below. In ga1 mutants that were generated in this way, we noted that ga1 seedlings (1.8%; n = 443) have the typical cotyledon defects of pin1 mutants (Figures 6A and 6B). Importantly, we noticed a strong increase in the number of such seedlings when we combined the ga1 and pin1 mutations (25%; n = 225 [of 900 ga1 pin1/PIN1 progeny seedlings]). Strikingly, the number of phenotypic seedlings exceeded the calculated sum (15.1%) for such seedlings from the single mutants (1.8% for ga1 and 13.3% for pin1). Since homozygous pin1 or ga1 pin1 seedlings can be obtained only as segregants from (ga1/ga1) pin1/PIN1 heterozygous parents, we confirmed the correlation of the cotyledon phenotypes with the ga1 and pin1 mutations by genotyping. Among 35 cotyledon-defective seedlings from the progeny of a ga1/ga1 pin1/PIN1 parent plant, we found that all but one of the tested seedlings was homozygous for the pin1 mutation (Figure 6C). We thus conclude that GA1 (and consequently GA biosynthesis) is needed for proper cotyledon development and suggest that GA1 and PIN1 interact genetically. Since we had observed that GA pathway mutants have decreased auxin transport (Figure 1) and that PIN protein levels are reduced in GA pathway mutants (Figures 2 and and3),3), we suggest that the cotyledon phenotypes observed in ga1 and ga1 pin1 double mutants are a consequence of defects in GA-dependent auxin transport regulation.

Figure 6.
GA Biosynthesis Is Required for Normal Seedling Development.

GA Is Required for Proper Root Gravitropism

We next tested the role of GA biosynthesis and signaling in root gravitropism, which requires the auxin efflux facilitator PIN2. Roots of pin2 mutants fail to establish a lateral auxin maximum that is formed in the root tip following gravistimulation, and pin2 mutants are fully agravitropic (Luschnig et al., 1998; Ottenschläger et al., 2003). When we examined gravitropic responses in ga1 mutant roots as well as in the three possible double mutant combinations of the three GID1 GA receptor genes, we found that each of the tested mutants is less gravitropic than the wild type (Figure 7A; see Supplemental Figure 4 online). To test whether the defect in gravitropic response is mediated by DELLA proteins, we also examined the ga1 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant, which lacks four of the five functionally redundant Arabidopsis DELLA repressors. In this analysis, we found that the partial agravitropism of ga1 roots is fully suppressed in the absence of DELLA protein function (Figure 7A), allowing us to conclude that proper root gravitropism requires GA biosynthesis and DELLA repressor–mediated GA signaling.

Figure 7.
Reduced Gravitropic Response in the GA Biosynthesis Mutant ga1.

Root gravitropic responses correlate with a redistribution of auxin in the root tip and the establishment of a new lateral auxin maximum, which can be monitored with the auxin (response) reporter DR5:GUS (Sabatini et al., 1999), allowing the visualization of the formation of an asymmetric GUS signal at the lower side of the gravistimulated root. We therefore examined DR5:GUS activity and distribution in root tips following gravistimulation of wild-type and ga1 mutant seedlings. Here, we found that the establishment of the new lateral auxin (DR5:GUS) maximum at the lower side of the root tip is significantly delayed in the ga1 mutant (Figures 7B and 7C). Whereas almost all of the tested wild-type seedlings (n ≥ 22) established the new GUS expression maximum within 2 h following gravistimulation, only 9 and 68% of the ga1 mutant seedlings (n ≥ 18) had established the lateral auxin maximum at 2 and 4 h after gravistimulation, respectively. Thus, the observed delay in gravitropic root bending in ga1 correlates with the reduction of PIN and PIN2 protein levels as well as with a delay in the PIN2-dependent lateral distribution of the DR5:GUS expression peak and auxin maximum.

DISCUSSION

In this study, we examine the contribution of GA to auxin transport and auxin transport–regulated growth and development in Arabidopsis. Our interest in the crosstalk of these two hormones was triggered by the observation that auxin transport is decreased in GA biosynthesis and signaling mutants from Arabidopsis (Figure 1). PINs are the major auxin efflux facilitators in Arabidopsis, and we show that the auxin transport defect of the GA pathway mutants and their auxin transport–dependent phenotypes, which we discuss below, can best be explained by a reduction of PIN protein levels (Figures 2 and and3).3). At the same time, we did not observe any changes in PIN protein polarity (Figures 2 and and4;4; see Supplemental Figure 2A online), and we therefore hypothesize that the reduction in auxin transport is the consequence of a reduction of PIN protein abundance rather than that of a change in the direction of auxin transport.

We also sought to understand the mechanisms that lead to a reduction of PIN protein levels in the GA mutants. To this end, we examined different PIN gene promoter GUS lines and their response to the lack of GA, either by introducing these transgenes into the ga1 background or by inhibiting GA biosynthesis with PAC (Figure 4). For PIN2 and PIN3, we did not observe any effects of GA on their expression in the root meristem where we had observed major GA-dependent changes in the accumulation of their protein products. For two PIN genes, PIN1 and PIN4, we found a GA-dependent decrease of PIN gene transcription, while PIN1 protein showed the opposite behavior (PIN4 was not tested in our study). It may thus be envisioned that the loss of PIN protein levels and the concomitant loss of auxin efflux leads, at least in the case of PIN1, to the activation of a compensatory transcriptional feedback mechanism. The existence of such compensatory transcriptional feedback mechanisms has already previously been postulated in a study that showed that the loss of individual PIN genes results in the transcriptional upregulation of other PINs (Vieten et al., 2005). In summary, our analysis indicates that the transcriptional regulation observed with the GUS reporters does not reflect the differences in PIN protein abundance that can be observed in the same backgrounds (Figures 2 and and3).3). Notably, our finding that GA is not a major positive regulator of PIN gene transcription is different from the role of GA previously reported in a study that examined the contribution of the DELLA protein RGA to the transcription of the AUX/IAA-type regulator SHY2, which is a transcriptional repressor of PIN gene expression, notably of PIN1, PIN3, and PIN7, in the transition zone of the root meristem of young seedlings (Dello Ioio et al., 2008; Moubayidin et al., 2010). There, it had been observed that the transcription of SHY2 is dependent on the presence and absence of the DELLA protein RGA. From this observation it was inferred that PIN gene transcription and PIN protein abundance, and consequently directional auxin transport, may be regulated in a DELLA- and GA-dependent manner in the transition zone of the root meristem (Moubayidin et al., 2010). We now show that in addition to this transcriptional regulation mechanism, which applies to the root transition zone, another regulatory mechanism exists that cannot be explained by transcriptional regulation. Since transcriptional regulation mechanisms could not explain the GA-dependent differences in PIN protein abundance, we turned to examining the behavior of PIN2:GFP. In these analyses, we observed a more pronounced increase in the vacuolar abundance of PIN2:GFP in ga1 mutants following ConA treatment, which blocks the acidification of the vacuole and thereby impairs vacuolar proteolysis. In agreement with this observation, we found a stronger increase in the abundance of PIN2 also in immunoblots of protein extracts prepared from root tips of ConA-treated ga1 seedlings. Importantly, the effect of ConA on PIN2 accumulation in ga1 mutants could be suppressed by GA treatment, indicating that the process can directly be modulated by the hormone. WM treatment, which blocks the vacuolar transport of PIN proteins, also led to a stronger increase in PIN2 protein levels in root tips. Our cell biological and immunoblot analyses therefore suggest that the differential GA-dependent accumulation of PIN proteins can at least in part be explained by increased PIN vacuolar targeting. Through which trafficking pathway PIN protein is targeted to the vacuole as well as which GA-dependent regulator controls PIN vacuolar targeting remains to be elucidated. Since we noted that the effect of GA on PIN accumulation is not immediate but requires several hours, we are tempted to speculate that PIN vacuolar targeting is controlled by an as yet unknown de novo synthesized transport regulator downstream from the GA signaling pathway.

In our studies, we also demonstrate a role for GA in two auxin transport–dependent processes, cotyledon development and root gravitropism, that, taken together, correlate with the reduction in auxin transport that we infer from the reduction of PIN protein levels that can be observed in GA pathway mutants. Defects in the proper initiation, positioning, and development of cotyledons are hallmark phenotypes of mutants deficient in auxin transport, such as pin1 (Galweiler et al., 1998), or auxin transport regulatory kinases, such as pid (Benjamins et al., 2001) and d6pk (Zourelidou et al., 2009). We observed several defects in cotyledon development in GA-deficient ga1 mutants (Figure 6A); however, they were seen only when care was taken to avoid the suppression of the GA deficiency of the ga1 mutant by omitting GA treatments after flower formation and during embryogenesis. As is normally the case for cotyledon development defects (Hadfi et al., 1998; Zourelidou et al., 2009; Huang et al., 2010), the penetrance of this phenotype in ga1 was relatively low but strongly increased in the presence of the sensitizing pin1 mutation (Figure 6). We thus conclude that GA is required in addition to PIN1 to promote proper auxin transport–dependent development during cotyledon formation. This establishes a previously unrecognized role for GA in cotyledon development with regard to auxin transport and auxin transport regulation. At least one previous study reports a cotyledon formation defect in a mutant with altered GA levels; however, this observation was not discussed in the context of auxin transport or auxin signaling (Singh et al., 2010). In that work, seedlings with altered cotyledon numbers were observed among the progeny of transgenic lines in which GA levels were reduced due to the misexpression of a GA catabolic GA2OXIDASE gene. Based on our observation that the ga1 mutant also has cotyledon formation defects and the correlation of this phenotype with altered PIN protein abundance, it may now be argued that the reduction of GA, and consequently PIN-mediated auxin transport, is causative for the seedling phenotypes observed in the previous study (Singh et al., 2010).

We further demonstrate that GA is involved in root gravitropism, a process that strictly requires the activity of PIN2 (Figure 7). Importantly, we find that GA contributes to, but is not strictly required for, root gravitropic responses. Since we also show that the reduced gravitropism of GA mutants correlates with a reduction of the formation of a new lateral auxin maximum in the root tip, we argue that GA regulates PIN protein levels and thereby promotes the redistribution of auxin that is required for root tip bending.

In summary, we propose that GA promotes developmental and growth processes that require auxin transport mediated by PIN auxin efflux facilitators (Figure 8). Based on this finding, it may be speculated that the growth defects of GA biosynthesis and signaling mutants are a result of altered auxin transport. However, experiments that would address the question of whether auxin transport defects repress growth at the whole plant level are difficult to perform since they may require the tissue-specific overexpression of different PIN proteins uncoupled from their posttranslational regulation mechanisms. Since our analysis of the root gravitropism phenotype suggests that these responses require GA as well as the suppression of DELLA repressor activity (Figure 7), we propose that the DELLA repressors may regulate the transcription of a gene required for PIN vacuolar targeting. The identification of such GA-dependent regulators will be the subject of further studies.

Figure 8.
Model of the Proposed Role of GA and GA Signaling in the Regulation of PIN Protein Abundance and PIN-Mediated Development and Growth Responses.

METHODS

Biological Material

ga1 (SALK_109115), ga1-3, ga1-3 gai-t6 rga rgl1-1 rgl2-2 (ga1-3 della), gai-1, gid1a-1 (SALK_044317) gid1b-1 (SM_3_30227) (gid1ab), gid1a-1 gid1c-2 (GABI_639F05) (gid1ac), gid1b-1 gid1c-2 (gid1bc), and pin1 (SALK_047613) mutants were previously described and characterized (Peng et al., 1997; Achard et al., 2006; Willige et al., 2007; Zourelidou et al., 2009). pin2 (SALK_042899) mutant lines (Arabidopsis thaliana Col) were identified in the SIGNAL database (http://signal.salk.edu/cgi-bin/tdnaexpress) and obtained from the Nottingham Arabidopsis Stock Centre. PIN1:PIN1:GFP, PIN2:PIN2:GFP, PIN3:PIN3:GFP, and AUX1:YFP:AUX1 were gifts from Jiri Friml, Christian Luschnig, Masao Tasaka, and Malcolm Bennett, respectively (Swarup et al., 2001; Benková et al., 2003; Abas et al., 2006; Zádníková et al., 2010). Transgenic lines expressing PIN1:GUS, PIN3:GUS, and PIN7:GUS were previously described (Vieten et al., 2005) and obtained from the Nottingham Arabidopsis Stock Centre, and PIN2:GUS and PIN4:GUS (Vieten et al., 2005; Abas et al., 2006) were gifts from Christian Luschnig. DR5:GUS was a gift from Tom Guilfoyle. Wild-type and mutant GA1 and PIN1 loci were identified by PCR-based genotyping using the primers 5′-CAGACCCGAGACAGTAACTGC-3′ and 5′-TCTCTACTCGAGGCAAGCTTG-3′ to test for GA1 and 5′-TCTCTACTCGAGGCAAGCTTG-3′ together with LBb1-3 to test for the T-DNA insertion in ga1 (SALK_109115); wild-type PIN1 was detected with the primers 5′-CAAAAACACCCCCAAAATTTC-3′ and 5′-AATCATCACAGCCACTGATCC-3′ and 5′-AATCATCACAGCCACTGATCC-3′ together with LBb1-3 to test for pin1 (SALK_047613).

ga1 mutant seeds were incubated at 4°C in 20 μM GA3 (ga1-3) and 100 μM GA3 (ga1 SALK_109115) for 5 d to induce germination. Seeds were then thoroughly washed five times in water and grown on standard growth medium (Murashige and Skoog [MS] medium/1% Suc). Adult plants were grown on standard soil mixtures. To rescue the phenotype of ga1 mutants, plants were sprayed repeatedly with 100 μM GA. GA treatment was omitted after flower formation when growing the ga1 and ga1 pin1/PIN1 mutants to be able to assess the contribution of GA biosynthesis to embryo development.

Auxin Transport Measurements

For auxin transport measurements, 25-mm stem segments were cut above the rosette of 6- to 7-week-old wild-type or mutant plants and placed for 30 min in an inverted orientation in 30 μL auxin transport buffer (500 pM IAA, 1% Suc, and 5 mM MES, pH 5.5). The stem segments were then transferred to auxin transport buffer containing 11 kBq (417 nM) radiolabeled [3H]IAA (GE Healthcare), with or without 100 μM NPA. After 2 h, the lowermost 5-mm segment was discarded because it was in direct contact with the radiolabeled [3H]IAA and 5-mm segments were dissected from the stem and macerated overnight in 300 μL Hydroxide of Hyamine 10-X (Packard Instrument Company). The solution was then neutralized by the addition of 300 μL acetic acid, and the uptake of [3H]IAA was quantified using a Wallac WinSpectral 1414 liquid scintillation counter (Perkin-Elmer Life Sciences). Three replicate measurements were performed for each genotype, and the experiment was repeated three times with reproducible results. The result of one experiment is shown.

Physiological Experiments

Gravitropic responses were measured in seedlings grown on vertically oriented plates containing MS medium/1% Suc. Seven-day-old seedlings were then transferred to the dark and kept in a vertical orientation for an additional 2 h. Subsequently, plates were reoriented by 135° and scanned after 6 h to determine their root angles using ImageJ software (NIH).

Cell Biology

For GUS staining of PIN:GUS lines, 7-d-old seedlings were fixed in heptane for 15 min and stained for 30 min (PIN2:GUS), 2 h (PIN3:GUS, PIN4:GUS, and PIN7:GUS), or 16 h (PIN1:GUS) with GUS-staining solution [100 mM Na-phosphate buffer, pH 7.0, 2 mM K4Fe(CN)6, 2 mM K3Fe(CN)6, 0.1% Triton X-100, and 1 mg/mL X-Gluc] and subsequently destained in 70% ethanol. For GUS staining of gravistimulated DR5:GUS transgenic lines, seedlings were incubated in GUS-staining solution [100 mM Na-phosphate buffer, pH 7.0, 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, 0.5% Triton X-100, and 1 mg/mL X-Gluc] for 16 h. GUS-stained seedlings were analyzed and photographed using a Leica MZ16 stereomicroscope with a PLAN-APOX1 objective (Leica). The number of seedlings with an asymmetric DR5:GUS maximum at the lower side of the root tip was quantified following GUS staining at 0, 2, 3, and 4 h after reorientation of 7-d-old seedlings to 135° (Figure 7B).

Fluorescence microscopy for identifying PIN:GFP and YFP:AUX1 distribution was performed using an Olympus BX61 fluorescence microscope with an XM10 digital black-and-white camera and a UPlanFL 4× objective (Olympus) or a FV1000/IX81 laser scanning confocal microscope with UAPO40XW3/340 (Figure 2A) and UPLSAPO60XW/1.2 objectives (Figures 2B, 5A, and 5B). Images were obtained and processed using the cellSens Dimension or FluoView software.

For PAC and GA treatments of wild-type seedlings (Col), 4-d-old seedlings were transferred to media containing 10 μM PAC and grown for an additional 2 d. The seedlings were then transferred and grown for 24 h on 10 μM PAC (PAC) without or with 10 μM GA3 (24 h GA). ga1 mutants were transferred after 6 d to medium containing 10 μM GA3 (or a solvent control) and analyzed after 24 h. For FM4-64 staining, seedlings were incubated in 1 μM FM4-64 (Invitrogen) for 10 min 5 to 7 h before microscopy. For ConA (1 μM) treatment, 5- to 8-d-old seedlings were grown in standard growth medium (MS medium/1% Suc) supplemented for 7 h with the inhibitor.

Protein Extraction and Immunoblotting

Total protein extracts were prepared in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10 μM MG132, 0.1 μM PMSF, and protease inhibitor cocktail [Sigma-Aldrich]). When detected from roots, nonspecific background bands were detected in PIN2 immunoblots from total protein extracts. To solve this problem, proteins were extracted from microsomal membrane fractions in this case. To this end, plant roots were homogenized in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM EDTA, 50 mM NaF, 10 mM10 NaVO3, 10 μM MG132, 0.1 μM PMSF, and protease inhibitor cocktail). Extracts were centrifuged for 5 min at 10,000g, and the supernatants were further centrifuged for 1 h at 100,000g in a Sorvall MTX 500 benchtop centrifuge (ThermoScientific). P100 Pellets were homogenized in 2× Laemmli and heated by 45°C for 5 min prior to loading onto the gels for immunoblotting. For ConA (1 μM) and WM (33 μM) treatments, 6-d-old seedlings were grown in standard growth medium (MS medium/1% Suc) supplemented with the inhibitors for 7 and 6 h, respectively. Anti-GFP (1:3000; Invitrogen), anti-PIN1 (1:4000; Nottingham Arabidopsis Stock Centre), and anti-PIN2 (1:3000; Agrisera) were used for immunoblotting. Coomassie Brilliant Blue–stained gels were used to control for equal loading.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are as follows: AUX1 (AT2G38120), GA1 (AT4G02780), GAI (AT1G14920), GID1A (AT3G05120), GID1B (AT3G63010), GID1C (AT5G27320), PIN1 (AT1G73590), PIN2 (AT5G57090), PIN3 (AT1G70940), PIN4 (AT2G01420), PIN7 (AT1G23080), RGA (AT2G01570), RGL1 (AT1G66350), and RGL2 (AT3G03450).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. YFP:AUX1 Protein Levels Are Not Obviously Altered in PAC-Treated Seedling Roots.
  • Supplemental Figure 2. GA Deficiency Does Not Affect PIN Protein Polarity and Does Not Alter Their Abundance in Differentiated Root Tissues.
  • Supplemental Figure 3. The Decrease in PIN:GFP Protein Abundance in ga1 Can Be Fully Suppressed by Long-Term GA3 Treatment.
  • Supplemental Figure 4. gid1 Mutants Impaired in GA Receptor Function Have a Reduced Gravitropic Response.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Anthi Katsiarimpa and Inês Barbosa for critical reading of the manuscript as well as various colleagues for sharing published and unpublished material. This work was supported by the following grants from the Deutsche Forschungsgemeinschaft to C.S.: SCHW751/6-1 (Arabidopsis Functional Genomics Network), SCHW751/7-1 (SPP1365), and SCHW751/8-1.

AUTHOR CONTRIBUTIONS

B.C.W., E.I., R.R., M.Z., and C.S. designed the research. B.C.W., E.I., R.R., and M.Z. performed the research. B.C.W., E.I., R.R., M.Z., and C.S. analyzed data, and B.C.W. and C.S. wrote the article.

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