Bloques de hormigón

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5.1 Introduction

TIP3 localise to both tonoplast and plasma membrane (PM), the two cellular endpoints of

the plant secretory pathway (Gattolin et al. 2011). Both TIP expression and localisation are

developmentally regulated during seed maturation and germination (Gattolin et al. 2011).

TIP3 has been visualised at both membranes by confocal microscopy from early torpedo stage embryos through to seed germination, although presence of TIP3 at the PM is

reduced in the drying seed (Gattolin et al. 2011). Dual localisation of TIP3 has also been

reported in developing pea cotyledons using immunological detection (Robinson et al.

1996).

The sixth (last) transmembrane domain and cytosolic tail of TIP3;1 was able to reach the tonoplast when fused to a signal peptide for translocation into the ER followed by a cytosolic bacterial enzyme (Hofte and Chrispeels 1992). Recently, we have found that

removing the distal C- terminal 23 amino acids of TIP3;2 (TIP3;2ΔC) abolishes PM

localisation (Gattolin, Carroll and Frigerio, unpublished). There must therefore be information in this final part of the TIP3 primary sequence, which is responsible for targeting to both tonoplast and PM. However, the specific sequence is yet to be elucidated, as is whether targeting to tonoplast and PM occurs simultaneously or whether the protein trafficked vectorially from PM to tonoplast.

Tonoplast proteins are thought to be co-translationally inserted into the membrane of the endoplasmic reticulum (ER) before being sorted through the secretory pathway to reach their destination via the Golgi-dependent or -independent routes (Jiang and Rogers 1998). TIP3;1 has already been reported to reach the tonoplast via the Golgi independent pathway

(Park et al. 2004). Park et al. (2004) showed in Arabidopsis protoplasts that TIP3;1

modified to contain the phaseolin C terminus N-glycosylation site, is not altered by Golgi resident glycan modifying enzymes until Brefeldin A treatment re-localises these enzymes to the ER. Brefeldin A (BFA) prevents the formation of COPI vesicles responsible for retrograde traffic from Golgi to ER, disrupting protein transport and causing Golgi proteins

to be retained within the ER (Nebenfuhr et al. 2002). This pharmacological tool has been

used to show that TIP3;1 traffics Golgi independently through its persistent tonoplast localisation in the presence of BFA. This has been shown both by immunodetection of the

protein in vacuole extracts from transformed tobacco protoplasts (Gomez et al. 1993), and

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hypocotyls (Rivera-Serrano et al. 2012). However to date, TIP3 trafficking has not yet

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5.1.2 Aims and Experimental Approach

- To identify whether targeting of TIP3 to the PM and tonoplast is as a result of dual

sorting or of a novel tonoplast trafficking route that uses PM as an intermediary station;

- to unravel the trafficking route by which TIP3 takes to reach these membranes.

Various TIP3-fluorescent protein fusions were visualised using confocal microscopy to confirm dual localisation and the role of the TIP3 C-terminus in PM localisation. Photoconversion, photobleaching and dexamethasone induced expression of TIP3- fluorescent tag fusions were employed as techniques to visualise dual or sequential targeting while BFA treatment was used to establish trafficking routes through the secretory pathway.

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5.1.3 Constructs

Figure 5.1 shows the diagrams of the constructs and the genetic background of the

Arabidopsis thaliana lines used in this Chapter. Photoconvertible proteins Dendra2 and mEoSFP were used as an approach to decipher order of TIP3 trafficking to the plasma membrane and tonoplast (as discussed in section 5.2.3). These particular photoconvertible proteins were available from a collaborator (Dr. Jaideep Mathur, University of Guelph), and so a tried and tested method was available. The mEoSFP-calnexin fusion was used as a

positive control given its reported success Mathur et al. (2010).

To investigate the Golgi dependent and independent pathways of TIP proteins, gnl1

background Arabidopsis lines were used. As described in sections 1.6.2 and 5.1, Arabidopsis GNL1 is insensitive to Brefeldin A (BFA), the drug used to disrupt trafficking via the Golgi. Therefore, using a knockout background renders these plants susceptible and

enables BFA treatment to disrupt this pathway. VHA-a1, the subunit of Arabidopsis V-

ATPase, is known to traffic to the TGN via the Golgi and is thus a suitable control for these experiments (Viotti et al. 2013).

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Figure 5.1: Schematic representation of the constructs used to produce the results in Chapter 5

Full genomic sequences for TIP3;1, TIP3;2, TIP1;1, Calnexin or VHA-a1 were cloned into pGreen-0029 with YFP or photoconvertible proteins Dendra2 or mEoSFP; apart from in the case of pDEX:TIP3;1-YFP where it was cloned into the Gateway pDEX vector (Dr Jens Steinbrenner and Professor Jim Beynon, University of Warwick). Some constructs

were used to transform gnl1 background plants in order to convey sensitivity to Brefeldin

A treatment. A list of constructs cloned previously to this body of work is listed in table 2.4. TP=Tonoplast, PM=Plasma Membrane, ER=Endoplasmic Reticulum, TGN=Trans Golgi Network. C ol -0 gnl 1

Genetic Construct Construct Expected Background Name Design Localisation

TP + PM TP + PM TP + PM TP TP + PM TP + PM ER TP + PM TGN TP + PM TP TP

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5.2 Results

5.2.1 TIP3 locates to tonoplast and plasma membrane independently of the position of the fluorescent protein tag

Dual localisation of TIP3 to the tonoplast and PM was shown to be independent of XFP

position in Gattolin et al. (2011). To confirm this is independent of YFP being fused to the

N or C terminus of TIP3;1, embryos expressing TIP3;1-YFP and YFP-TIP3;1 were imaged by confocal microscopy (figure 5.2A-B). It is difficult to capture the PM by confocal microscopy in these embryos during imbibition, as amount of TIP3;1-YFP or YFP-TIP3;1 at the tonoplast is so high that signal becomes saturated at a low laser power. At this low laser power visualising the signal at the PM, which is approximately 10-fold lower in fluorescent signal, is problematic. Nevertheless, in combination with the results from

Gattolin et al. (2011) (C-D), it is clear that dual localisation is maintained regardless of the

position XFP is fused to.

From these results, it is deducible that XFP does not mask an essential sorting signal causing TIP3 to be redirected. In addition, PM localisation has already been found not be an artefact of overexpression by expressing tip3;2pro:TIP3;2-YFP in TIP3;2 tDNA

knockout lines mutant lines (tip3;2) (Gattolin et al. 2011). Furthermore, use of the TIP3;2

promoter to drive expression of YFP-TIP1;1 in tip3;2 has confirmed that PM localisation

is due to a sorting signal contained within the TIP3 sequence itself, as this construct results

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Figure 5.2: TIP3;1 and TIP3;2 translationally fused to YFP localises to the tonoplast and plasma membrane independently of N or C terminal position

(A) TIP3;1-YFP and (B) YFP-TIP3;1 expressed under their native TIP3;1 promoters are

localised to the PM and tonoplast in mature embryos. These images are of embryo cotyledons which have been imbibed for 24 hours. Image B was acquired at high detector

gain in order to visualise PM, resulting in several overexpressed fluorescent structures. (C)

TIP3;2-YFP and (D) YFP-TIP3;2 expressed under their native TIP3;2 promoters are also

localised to PM and tonoplast in mature embryos (C-D taken from Gattolin et al. 2011).

Scale bars = 10µm.

TIP3;1-YFP YFP-TIP3;1

A B

TIP3;2-YFP YFP-TIP3;2