Trafficking SNARE SYP132 Partakes in Auxin-Associated Root Growth^,

Abstract

Auxin regulates abundance of the trafficking SNARE SYP132 over the time course of root growth and gravitropic responses.

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In plants, cellular membrane traffic coordinates osmotic cell expansion and ion transport for cellular homeostasis and growth (Karnik et al., 2017). The plant hormone auxin regulates growth responses in roots (Haswell, 2003; Enami et al., 2009; Monshausen et al., 2011; Ichikawa et al., 2014); even so, little is understood about auxin-mediated control of membrane traffic in this tissue. Recently we identified as a target for hormone regulation a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein, the Syntaxin of Plants 132 (SYP132). SYP132 is a member of the superfamily of proteins mediating vesicle fusion. Auxin modulates SYP132 transcript levels and SYP132 protein expression to influence membrane traffic associated with this SNARE (Xia et al., 2019). Unusual for a secretory SNARE, SYP132 is involved in the endocytosis of plasma membrane H⁺-ATPase proteins and reduces their density and activity at the plasma membrane, thereby suppressing H⁺ transport in the shoots (Xia et al., 2019). In roots, the SNAREs SYP132 and SYP123 are both essential to the plant. SYP123 accumulates at the growing root hair tip. By contrast, SYP132 distributes evenly throughout the root as well as the shoot, suggesting that this SNARE has a constitutive role in growth (Enami et al., 2009; Ichikawa et al., 2014).

We explored SYP132 in root elongation and auxin-dependent growth responses in Arabidopsis (Arabidopsis thaliana) taking advantage of tools developed previously (Xia et al., 2019). We conclude that, in roots, auxin leads to an initial decrease, followed by an increase in SYP132 protein expression. Under gravitropic stimulation, this temporal behavior closely matches the second phase of differential auxin-induced cell expansion, which drives root bending (Guilfoyle et al., 1992; Band et al., 2012). We also observed that SYP132 over-expression masked the dose-dependent response in root growth to auxin and as in shoots (Xia et al., 2019), it also reduced root growth. In gravistimulated roots, SYP132 expression was differentially regulated at the top and bottom flanks of the organ. As such, SYP132 protein expression has an inverse correlation with cell growth. Thus, we identified that the SNARE SYP132 and its regulation by the hormone auxin are associated with growth responses in roots.

Auxin Regulates SYP132 Expression and Influences Root Growth

Auxin differentially regulates SYP132 expression in shoot and root tissue (Xia et al., 2019). In shoots, auxin reduces SYP132 expression and endocytic traffic associated with this SNARE, thereby increasing PM H⁺-ATPase density and promoting shoot growth. To determine how auxin affects SYP132 expression in roots, we used Arabidopsis seedlings expressing GFP-fused SYP132 under the SYP132 native promoter (SYP132p::GFP-SYP132; Ichikawa et al., 2014). Auxin analog 1-Naphthalene-acetic acid (NAA) was used in these experiments as it is stable and efficiently penetrates cells (Delbarre et al., 1996). Seedlings were grown on half-strength Murashige and Skoog (0.5× MS) media and infiltrated with auxin (NAA, 10⁻⁶ M) or 0.02% (v/v) ethanol in water (control). Roots were separated from shoots and sampled at different time points after auxin treatment for immunoblot analysis using anti-GFP antibodies (Fig. 1A). We found, following auxin treatment, that SYP132 protein levels initially declined over the first 30 min of auxin treatment, but within 60 min SYP132 expression recovered and, after 240 min, SYP132 levels were higher compared with the control (Fig. 1, A and B). GFP-SYP132 protein quantity was calculated for each time point using densitometric quantification of bands and normalized to the total protein per lane (Fig. 1B).

Figure 1.

Auxin regulates SNARE SYP132 abundance during root growth. A, Immunoblot analysis of separated root tissue following treatment with 10⁻⁶ M NAA (auxin) for up to 240 min. Seedlings were grown on 0.5× MS plates for 3 weeks before treatment. Total protein was resolved on a single gel (10%, SDS-PAGE), and immunoblots were performed using anti-GFP (Abcam) to visualize GFP-SYP132 (∼62 kD, top) and antiactin (Agrisera) to detect reference protein actin (∼45 kD, middle). Black arrows indicate the expected band position. Ponceau S staining (bottom) was used to detect total protein on the membrane. B, Graph showing relative change in GFP-SYP132 protein expression compared with 0.02% (v/v) ethanol in water (control), calculated by densitometric analysis of immunoblots using Fiji software and normalized to total protein in each lane. Data are mean ± se; n = 3 experiments. C, Main root lengths of 3-d-old wild type and SYP132-OX Arabidopsis seedlings. Data are mean ± se, relative to wild type. Asterisk indicates statistical significance using ANOVA (*P < 0.05), n ≥ 300. D, Root growth rates in wild type and SYP132-OX seedlings in response to treatment with auxin at different concentrations. Seedlings were germinated on 0.5× MS plates, and after 5 d they were transferred to 0.5× MS plates containing 0 (control), 10⁻⁹, 10⁻⁸, 2.5 × 10⁻⁸, and 10⁻⁷ M NAA (auxin). Main root growth rates, in millimeters per hour, were calculated over 48 h. Data are mean ± se (n ≥ 50 roots, from three independent experiments). Statistical significance using ANOVA is indicated by letters (P < 0.001). E, Representative images of Arabidopsis seedlings at 0 and 48 H, as described in D. Scale bar = 1 cm. F, Immunoblot analysis using anti-RFP (top) to detect RFP-SYP132 (∼61 kD) protein expression in the Arabidopsis SYP132-OX line. Ponceau S staining of Rubisco was used as loading control (bottom).

Exogenous auxin treatment is known to suppress root growth in a dose-dependent manner (Rahman et al., 2007). To test how SYP132 influences root growth, we analyzed main root length. In the absence of a viable homozygous syp132 mutant (Park et al., 2018), root growth in Arabidopsis lines constitutively overexpressing SYP132 (SYP132-OX, 35S::RFP-SYP132, Xia et al., 2019) were compared against wild-type plants. Seedlings were grown on 0.5× MS plates for 5 d before measurements (Fig. 1C). We found that SYP132-OX seedlings had shorter root lengths compared with the wild type (Fig. 1C). We also tested the effect of SYP132 in auxin-associated root growth. Arabidopsis seedlings grown on 0.5× MS plates were transplanted to plates supplemented with increasing concentrations of NAA, and root elongation rates were measured over 48 h (Fig. 1, D–F). In wild-type Arabidopsis roots, 10⁻⁹ M NAA had no significant effect on root growth, but as the NAA concentration was raised (10⁻⁸ M and 2.5 × 10⁻⁸ M NAA), root growth rates decreased. We carried out parallel measurements with SYP132-OX roots. As expected, in the absence of auxin supplementation, we observed reduced elongation rates compared with the wild-type plants. However, increasing NAA had no additional effect on root growth even at a higher concentration range of 10⁻⁸ and 2.5 × 10⁻⁸ M (Fig. 1, D and E). Very high auxin (10⁻⁷ M NAA) severely inhibited root growth. We noted that auxin did not alter RFP-SYP132 protein levels in SYP132-OX plants (Xia et al., 2019), which discounts the possibility that auxin regulates the SNARE through additional pathways such as degradation. Instead, these observations are most easily explained as a result of saturation of SYP132 expression and masking of the auxin dose-dependence in root growth in the SYP132-OX plants (Fig. 1, D and E). Regardless of the interpretation, however, these findings support the involvement of SYP132 in auxin-mediated root growth responses.

SYP132 Contributes to Differential Root Growth Responses and Gravitropic Curvature

Auxin regulates plant gravitropic root growth responses through a complex set of molecular mechanisms (Su et al., 2017). Upon gravistimulation (see schematic, Fig. 2A), auxin gradients are generated across the top and bottom flanks of the root, which drive root growth responses. Auxin levels are higher on the bottom flank of the root, where cell elongation is reduced, compared with the top flank, and these differences in growth result in curvature of the root toward the Earth’s gravity vector (Swarup et al., 2005; Monshausen et al., 2011; Velasquez et al., 2016; Barbez et al., 2017; Su et al., 2017).

Figure 2.

Auxin regulates SNARE SYP132 abundance during root gravitropism. A, Schematic representation of gravitropic stimulation of root tips. B, Fold changes in SYP132 transcript levels in gravistimulated root tips at different time points. Wild-type seedlings were grown vertically on 0.5× MS media for 6 d after which the plates were turned at an angle of 135° and root tips were excised and collected at 0, 10, 30, 120, and 240 min after gravistimulation. Fold changes in gene expression with reference to time 0 were calculated as 2^-ΔΔCT (Livak and Schmittgen, 2001). The mitochondrial 18S rRNA gene (AtMg01390) was used as a control. Asterisk indicates statistical significance calculated using ANOVA (*P < 0.050); n = 3. C, Representative confocal images of the midsection of SYP132p::GFP-SP132 Arabidopsis root tips, gravistimulated (135°) for 0, 30, 120, and 240 min. Boxed areas represent parts of the elongation zone used for fluorescence analysis. Arrow represents the direction of the gravity vector. Scale bar = 50 μm. D, GFP-SYP132 expression in top and bottom flanks of gravistimulated roots marked in (C). Data are mean of fluorescence intensities measured using Fiji software. Asteriks indicate statistical significance between each top and bottom flank values is calculated using ANOVA (*P < 0.001 and **P < 0.001); n = 6. A.u., arbitrary units. E, Mean cell length ratios for bottom and top flanks of the roots ± se. Length of cells on opposite ends of gravistimulated roots represented in (C) were measured from confocal images using Fiji. Statistical significance using ANOVA is indicated by letters (P < 0.001); n ≥ 20. F, Schematic representation of SYP132 protein levels (bold lines) in top (red lines) and bottom (black lines) flanks and corresponding changes in growth (dashed lines), referenced from cell lengths in gravistimulated roots over 240 min. Schematic represents data in (C) to (E). G, Gravitropism diagram representing distribution of root curvature angles following 6 h of gravistimulation, each assigned to one of eight 45° sectors. The lengths of the bars in the diagram represent the percentage of wild type, SYP132-OX, and syp132^T seedlings occurring in each assigned sector. H, Images from the same wild type, SYP132-OX, and syp132^T seedlings following gravistimulation, as in (A), acquired at different time points. Images are representative of n = 5 independent experiments. Bar = 5 mm. Hr, hour. I, Root curvature angle after gravistimulation over 6 H, depicting reoriented root tip growth toward the Earth’s gravity vector. Data are mean ± se (n ≥ 50 roots, from three independent experiments). Asterisk indicates statistically significant differences for each time point compared with the wild type using ANOVA (*P < 0.001). J, Bar graph depicting growth rate of roots after gravistimulation, measured over 24 h. Data are mean ± se. Letters indicate statistical significance using ANOVA (P < 0.050); n ≥ 30 roots, from three independent experiments.

To test the role of the SNARE SYP132 in gravitropic growth, a gravistimulus was applied to wild-type Arabidopsis roots by rotating plants grown on plates to 135° (Fig. 2A). It is not possible to effectively segregate tissue from the top and bottom flanks of the root tip. Therefore, total root tip tissue was collected at different time points and was analyzed for SYP132 transcript levels by reverse transcription quantitative PCR. During phase 1 of the gravitropic response, an auxin gradient is generated across the root tip and initiates auxin-regulated transcriptional changes and differential growth. Subsequently, auxin asymmetry is lost. Phase 2 follows after approximately 100 min during which the downstream targets or effectors continue to contribute to growth for gravitropic curvature of the root in the absence of an auxin gradient (Band et al., 2012). We observed SYP132 expression in gravistimulated root tips to follow this second phase of growth (Fig. 2B). During the first 30 min after stimulus, total root tip SYP132 transcript levels did not change, but after 120 min, total root tip SYP132 transcripts were higher relative to those at the start of the stimulus (Fig. 2B). Such an increase in total root tip SYP132 levels would underestimate local increase of the SNARE within regions of the root tip.

To understand how changes in SYP132 transcripts correlate with SYP132 protein distribution, confocal imaging of gravistimulated SYP132p::GFP-SP132 Arabidopsis root tips (Ichikawa et al., 2014) were performed. The GFP fluorescence intensity in root cells lining the top and bottom flanks of the root elongation zone was measured at different time points (Fig. 2C). GFP-SYP132 fluorescence intensity was similar in the top and bottom flanks of the roots at 0 and 30 min after stimulus, but after 120 min, GFP-SYP132 fluorescence at the top flank was reduced compared with the bottom flank (Fig. 2D). This unequal distribution of GFP-SYP132 complemented the differential in cell expansion determined by changes in the cell-length ratio for cells lining the lower flank compared with those lining the top flank (Fig. 2E). Differential in SYP132 expression aligns inversely with growth in the top and bottom flanks of the root (Fig. 2F).

Finally, to explore the physiological role of SYP132 in gravitropic growth, we measured the angle of curvature and growth rates of gravistimulated root tips in wild-type, SYP132-OX, and heterozygous SYP132 transfer (T) DNA insertion mutant (syp132^T) Arabidopsis seedlings (Fig. 2, G–J), of which the latter have a 5-fold reduction in SYP132 expression (Xia et al., 2019). When compared with the wild type, the SYP132-OX lines showed increased gravitropic curvature and a reduced root growth rate over 24 h. Conversely, in the heterozygous syp132^T mutants, gravitropic curvature was reduced, but root growth rate was higher (Fig. 2, G–J). We understand the difference between the wild type, SYP132-OX, and syp132^T mutants in gravistimulated roots assuming that auxin evokes finite changes in SYP132 expression. Key to this interpretation is a recognition that SYP132 suppresses cell elongation, thus reducing the rate of growth. Gravistimulation evokes radial auxin gradients that associate with a differential in SYP132 expression across the top and bottom flanks of the root tip. Where SYP132 expression increases, growth is reduced. Overexpressing this SNARE elevates background SYP132 protein abundance and reduces overall growth. It follows that in the SYP132-OX line, the differential in growth is enhanced and gravitropic curvature is more pronounced. Conversely, in the syp132^T mutant, background SYP132 protein abundance is lower, the overall growth rate is higher, and there is less curvature (Fig. 2, H–J).

There is evidence for auxin in apoplastic pH regulation mediated through plasma membrane H⁺-ATPase (Fasano et al., 2001; Barbez et al., 2017). We suggest that these same features may be responsible for the asymmetric ion fluxes that drive root cell expansion and gravitropism. Our data align with the role of SYP132 and associated traffic on plasma membrane H⁺-ATPase endocytosis and consequent decrease in function of the proton pump in the shoot tissue (Xia et al., 2019). In conclusion, we propose that auxin regulates the SNARE SYP132 during root growth and gravitropic responses and that hormone-regulated membrane traffic associated with this SNARE is important for understanding of auxin-associated growth.