In the DMSO control, GFP-TIP2;1 and TIP3;1-YFP accumulated in the vacuolar membrane and they could be visualized as a smooth surface in cortical sections of the cell or as continuous membrane in medial sections (Figure 3A). The localization of TIP1;1-YFP was stronger in vacuolar bulbs, but it could still be detected in the tonoplast as expected (Figure 3D, E). When plants were treated with C834, both GFP-TIP2;1 and TIP3;1-YFP accumulated in the ER network (Figure 3F, G), whereas TIP1;1-YFP was found in the tonoplast and vacuolar bulbs in the same patterns as the control (Figure 3H, I). Because C834 affected trafficking of both TIP3;1
Figure 2. GFP-TIP2;1 accumulates in the Endoplasmic Reticulum in C834-treated cells. Three-day-old seedlings expressing GFP-TIP2;1 and mCherry-HDEL were exposed to DMSO (control, A, B) or 55 uM C834 (C, D) for 48 h, and imaged under a confocal microscope. Signals from GFPTIP2;1 (green), mCherry-HDEL (red) and merged image are shown. Scatter plots showing pixel intensity in the red (Y axis) or green (X axis) channel for each image is also shown. While signal in the DMSO treated cells are distributed equally (A, B), the pixels in the C834-treated cells show strong correlation between the two channels (C, D). Both cortical (A, C) and medial (B, D) sections of the same cells are shown. Bar = 10 mm.
and GFP-TIP2;1 markers, but had no effect on TIP1;1, it was defined as Class I (Table 1, Figure 3). C410 and C755 induced the accumulation of all three tonoplast markers to the ER network and were defined as Class II (Table 1, Figure S4). C103 and C578 affected all three markers (Figure S4), and also disrupted the appearance of the ER marker by either forming vesiculated structures (Figure 1E) or inducing bright cytoplasmic staining (Figure 1F). These two probes were defined as Class III (Table 1), and they represent broader inhibitors of the endomembrane system compared to Class I and II. Our finding that the Class I compound C834 affected GFP-TIP2;1 and TIP3;1-YFP, but not TIP1;1-YFP indicated that the former are trafficked via similar mechanisms, but the latter utilizes a different pathway that isinsensitive to C834. Here, we describe the bioactivity of C834 in more detail (Table S1).Multiple trafficking pathways are insensitive to C834
In order to understand the bioactivity of C834, we first determined the minimum concentration and incubation time for bioactivity, as well as its reversibility and its effect on plant growth. It was determined that C834 induced the accumulation of GFPTIP2;1 at the ER as early as 8 h after treatment with 55 mM in roots, but no effects were observed in cotyledons or hypocotyls even when plants were treated at 110 mM for 48 h. Possible reasons for this result are that a C834-target is not expressed in shoots or that its inactivation is overcome by the expression of a functionally redundant protein. The effects of C834 on traffickingthat, at least for the markers analyzed, the effect of C834 on trafficking was specific to a subset of tonoplast membrane proteins. Plants grown in the light in the presence of C834 showed a significant (,40%) reduction in primary root elongation when compared to the control, but had normal seedling phenotype (Figure 4A). Eight-day old wild type plants grown in media containing DMSO (control) had roots of 1.7+/20.07 cm, but those grown in the presence of 55 mM C834 were 1.08+/ 20.07 cm. Root hair elongation was dramatically suppressed by C834 treatment (Figure 4B, C), possibly due to the critical roles of the endomembrane trafficking on root hair growth [52]. Overall, these results indicate that C834 had mild effects on seedling development, except for root and root hair elongation, which were dramatically affected.
Figure 3. C834 uncouples two pathways for tonoplast proteins trafficking. Three-day-old seedlings expressing GFP-TIP2;1 (A, F), TIP3;1-YFP (B, C, G) or TIP1;1-YFP (D, E, H, I) were exposed to DMSO (control, A) or 55 mM C834 (F) for 48 h and imaged under a confocal microscope. ER network localization (arrows) was observed in C834treated GFP-TIP2;1 and TIP3;1-YFP seedlings, but not in TIP1;1-YFP. Cortical sections are shown in B, D and F-H. Medial sections are shown in A, C, E and I. Arrowheads indicate vacuolar “bulbs” labeled with TIP1;1-YFP. Bar = 20 mm. TIP2;1 and TIP3;1 traffic to the tonoplast via a BFAinsensitive pathway
The trafficking of TIP3;1 in tobacco and Arabidopsis leaf protoplasts occurs via a Golgi-independent mechanism and is BFA insensitive [17,22]. We hypothesized that GFP-TIP2;1 also trafficked to the vacuole via a BFA-insensitive pathway, which we propose is the target of C834, but TIP1;1 utilized a BFAsensitive, C834-insensitive pathway similar to the one described for a BP-80 chimeric protein [17]. The fact that neither TIP3;1 [22] or GFP-TIP2;1 (data not shown) are glycosylated in Arabidopsis prevented us from examining the presence or absence of Golgidependent glycosylation in these proteins. As an alternative, we tested this hypothesis by examining the effects of BFA, an inhibitor of ER-to-Golgi vesicle trafficking [53], on TIP protein localization.
in root cells were reversible after wash-out (Figure S5A), indicating that the C834 target was not irreversibly modified. We noticed early on that C834 inhibited trafficking only after the media had been incubated in the light (Figure S5D-F). Therefore, all the experiments presented here were done with light-activated C834 media. In order to determine the specificity of the trafficking pathways affected by C834 in more detail, we tested the effect of this inhibitor on the localization of multiple post-Golgi markers. First, we tested the effect of C834 on other tonoplast proteins including TRANSPARENT TESTA12 (TT12)-GFP [40] and VAMP711-YFP [37]. Neither TT12-GFP nor VAMP711-YFP were affected by C834 (Figure S6), indicating that these proteins are likely to be trafficked via a similar pathway as TIP1;1. Markers to other compartments were tested to determine the effect of the inhibitor on the endomembrane system as a whole. These represented Aleu-GFP for vacuole lumen [42], SYP32-YFP as marker for Golgi [37], SNX1-GFP as marker of the PVC/MVB [41], and YFP fusions to the endosomal markers RabG3f, RabA5d, and RabC1 [37]. Using this approach, we did not detect any differences in the localization of any of these markers between the DMSO and C834 treatments (Figure S6) indicatingFigure 4. C834 is a strong inhibitor of root hair elongation. Plants were grown in the light in media containing DMSO or 55 mM C834 for 7 days prior to imaging. Bar = 5 mm (A) or 1 mm (B).
