Since it was not possible to study targeting of TIP3;1 using photoconvertible proteins or
photobleaching, an ad hoc induction system of TIP3;1-YFP was devised. Using the pDEX
Gateway plasmid, (Dr Jens Steinbrenner and Prof Jim Beynon, University of Warwick) expression of the inserted gene can be controlled by dexamethasone (DEX) treatment. This pDEX Gateway vector is derived from the DEX inducible plant promoter vector pTA7001 originally described by Aoyama & Chua (1997). The plasmid contains a transacting synthetic GVG transcription factor, which is expressed under the control of the cauliflower mosaic virus 35S promoter and flanked by the polyadenylation signal of the pea ribulose bisphosphate carboxylase small subunit rbcS-E9. Using Gateway cloning, the sequence for TIP3;1-YFP was inserted into the vector, flanked by the polyA sequence of the pea rbcS- 3A and under the control of a cis- acting element containing 6 Gal4 upstream activating
sequences (UAS) and the -46 to +1 region of the 35S promoter (Aoyama et al. 1997).
Figure 5.9 illustrates this setup schematically.
The constitutively expressed synthetic GVG transcription factor contains the hormone binding domain (HBD) of vertebrate glucocorticoid receptor, the transactivation domain of herpes virus VP18 and the DNA binding domain of yeast Gal4 TF. In the absence of its ligand DEX, interactions of the GR-HBD with cytosolic chaperones hold GVG in the cytosol. In the presence of DEX, GVG can enter the nucleus. The Gal4-DNA binding domain of GVG interacts with the Gal4 UAS elements of the promoter activating transcription of the inserted gene; TIP3;1-YFP.
The induction system was initially tested in stably transformed Arabidopsis thaliana
seedlings. Testing the system in these tissues enabled us to explore methods and time course of induction as well as concentration of DEX required, without potential complication of the effects of germination on induction or the potential negative effects of
DEX on induction on germination. Seedlings germinated and grown on ½ MS agar plates
for five days were transferred to plates supplemented with 1µM (Bhorgi et al. 2010) or
30µM DEX (Lewis, 2012). After 24 hours, strong TIP3;1-YFP signal was detected in seedlings subjected to 30µM DEX (figure 5.10 A-B). Fluorescence of seedlings from these plates was significantly brighter than those exposed to 1µM DEX, indicating that induction of TIP3;1-YFP expression is responsive to DEX concentration. From this point onwards, a 30µM DEX concentration was used.
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Figure 5.9: TIP3;1-YFP expression is induced using a hybrid transcription factor ‘GVG’ as activated by Dexamethasone.
To induce TIP3;1-YFP expression, the translational fusion was cloned into the pDEX Gateway vector (Jens Steinbrenner and Jim Beynon, University of Warwick). This vector is based on the pTA7001 binary vector as outlined in Aoyama and Chua 1997. The ‘GVG’ hybrid transcription factor, comprised of the DNA-binding domain of the yeast transcription factor GAL4, transactivating domain of the herpes viral protein VP16 and HBD of the rat glucocorticoid receptor (GR), is constitutively expressed. In the presence of dexamethasone (DEX), GVG can enter the nucleus and activate expression of TIP3;1-YFP by binding to GAL4 UAS fused to a small region of the CaMV 35S promoter (TATA).
Adapted from Aoyama and Chua 1997.
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Figure 5.10: TIP3;1-YFP expressed under dexamethasone-inducible promoter localises to the ER and tonoplast in 7 day old seedlings.
5 day old seedlings germinated and grown on ½ MS 0.8% agar plates were transferred to plates supplemented with 30µM dexamethasone (DEX) for 24 hours. (A) After 24 hours of exposure to DEX, TIP3;1-YFP was clearly visible using a stereomicroscope in comparison to (B) wild type. (C-H) Confocal microscopy confirmed the localisation of TIP3;1-YFP to the tonoplast, as indicated by transvacuolar strands (*) and (G) ER but not (D-E, G-H) the PM as there is no colocalisation with FM4-64 styryl dye, as shown with arrows. Green is TIP3;1-FP and Red is autofluorescence in (A-B) and FM4-64 PM dye in (D-H). Scale bars = 10µm ^ * * * * A B A B R adi cl e C ot yl edon
DEX-induced Col-0 YFP YFP + FM4-64
* * ^ ^ ^ ^ ^ C D E F G H
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After two days of DEX exposure, seedlings were imaged by confocal microscopy to study TIP3;1-YFP cellular localisation. TIP3;1-YFP was visualised at the tonoplast in both root and cotyledons, as indicated by the presence of transvacuolar strands (figure 5.10 C and F), transvacuolar strands highlighted with asterisks). These transvacuolar strands could be confirmed in future experiments by using GFP-fABD2, a marker line for actin. In this line, GFP is fused to the sequence for ABD2 (actin binding domain 2) of the Arabidopsis fimbrin AtFIM1, which binds to actin filament bundles (Sheahan et al. 2004). Actin forms part of the structure of transvacuolar strands. The styryl PM dye FM4-64 was used to delineate the plasma membrane (figure 5.10 D-E, F-H, with arrows) from the tonoplast. The lack of consistent colocalisation at the cell perimeter confirms that TIP3;1-YFP is localised only to the tonoplast in these induced seedlings. Orange patches (figure 5.10D) can be explained by the fact that FM4-64 is endocytosed from the PM and can subsequently be incorporated into other organelle membranes, including the tonoplast. These observations match our previous observation that dual localisation of TIP3 to PM and tonoplast occurs only in maturing and germinating embryos.
Once the DEX concentration and time course of induction were established, sterilised dry seed of the same transgenic line were plated directly onto ½ MS agar containing 30µM DEX. This would allow us to observe TIP3;1-YFP localisation at a developmental time point when dual localisation to the tonoplast and PM is expected. Initial observations indicated that seeds did not fluoresce until after radicle emergence from the seed coat, roughly 48 hours following imbibition. At this point, TIP3;1-YFP is localised solely within the endoplasmic reticulum (ER), just as the PSV are in transition to becoming LV (figure 5.11 A-D). Once LV are formed, and take up most of the cellular space, the tonoplast lies directly adjacent to the PM, making it very difficult to distinguish clearly between the two. In addition, post-germination, TIP3 is not targeted to the PM. Imaging beyond this developmental time point is therefore uninformative, yet necessary in order to determine where TIP3;1-YFP localises following exit from the ER.
In order to decrease the time taken for TIP3;1-YFP induction to occur, and maximise the window for observation of targeting, dry embryos were removed from their seed coat and placed on ½ MS agar 30µM DEX to imbibe and induce. Less than 24 hours after plating, TIP3;1-YFP was expressed in embryos and localised to the ER (figure 5.11 E-F). This ER localisation was maintained for almost 72 hours following this observation, at which point PSV transition to LV and tissues develop into that of a germinated seedling.
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Throughout the window of observation for these different time points, embryos were continually incubated on ½ MS agar 30µM DEX. In some places large aggregates of fluorescent protein were apparent, which could be an indication of stress. In addition, it was thought that TIP3;1-YFP may be retained in the ER as a result of continual exposure to DEX and therefore stress caused by excessive expression.
To test if retention of TIP3;1-YFP in the ER was a result of stress by prolonged exposure to DEX, embryos extracted from seed coats and plated directly onto ½ MS agar 30µM DEX were transferred to ½ MS agar-only plates 24 hours later, which is when TIP3;1-YFP expression has been induced. TIP3;1-YFP fluorescence was still clearly visible by stereomicroscopy 24 hours after transfer, however TIP3;1-YFP was still observed only at the ER (figure 5.11 G-H). 45 hours after transfer, fluorescence had substantially decreased and a low signal of YFP was observed to be maintained at the ER. Further to this observation, a small number of aggregates were once again observed; the Frigerio group has observed similar structures to these in numerous live embryo or seedling samples subjected to extended observation time by confocal microscopy. Whilst the identity of these structures is unknown, these structures may be an artefact of prolonged exposure to the excitation laser during imaging.It can therefore be concluded that stress to the embryo from prolonged exposure to DEX, is not responsible for the retention of TIP3;1-YFP in the ER.
In order to determine whether TIP3;1-YFP does eventually leave the ER, a time frame of at least 72 hours is required from the point of induction. To ensure PSV and PM are sufficiently spatially separated, it is necessary to image developing, rather than mature embryos found in the dry seed (Bowman and Mansfield 1993). Excising mature green embryos from their developing seed coat, and plating them on ½ MS agar 30µM DEX was found to induce expression of TIP3;1-YFP embryos within 24 hours. With this knowledge, embryos from even earlier developmental stages than the mature green embryo stage will be induced and observed. Figure 5.12 outlines which main stages of Arabidopsis embryo development have been used so far, and those that will be used in future experiments.
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Figure 5.11: TIP3;1-YFP expressed under a dexamethasone inducible promoter localises to the ER in germinated seedlings and mature embryos.
(A-D) Dry seeds were plated on media supplemented with 30µM dexamethasone (DEX) and imbibed for 48 hours. Once seeds had germinated, TIP3;1-YFP was localised to ER. (E-F) Embryos extracted from seed coat after 1 hour imbibition were plated on media containing 30µM DEX. Between 21 and 70 hours after exposure to DEX, TIP3;1-YFP was localised to ER in embryos. (G-H) Embryos which were exposed to 30µM DEX for 20 hours, and subsequently transferred to media without DEX, expressed TIP3;1-YFP for a further 48hours where it was localised at the ER. Green is TIP3;1-YFP, blue is autofluorescence of PSVs and red is PM FM4-64 dye. Scale bars = 10µm
Se ed s Em b ry o s Em b ry o s re m ov ed f ro m DE X Cotyledon Radicle A B C D E F G H
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Figure 5.12 TIP3;1-YFP expression will need to be induced by DEX in early
developmental stages of Arabidopsis thaliana embryos
Copied from eFP browser (Winter et al. 2007), the developmental stages of A. thaliana embryo development and their respective silique sizes. The red arrow indicates the earliest stages of embryo development which were used in these experiments. Since TIP3 is expressed from late torpedo stage, embryos from earlier developmental stages, indicated with the green bar, will be used in future experiments. This will enable imaging of the PSV for a longer period of time.
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