INVITED REVIEW Blackwell Publishing, Ltd. GFP is …fb/Brandizzi et al. JMic.pdfOur understanding of...

© 2004 The Royal Microscopical Society

Journal of Microscopy, Vol. 214, Pt 2 May 2004, pp. 138–158

Received 22 October 2003; accepted 22 December 2003

Blackwell Publishing, Ltd.

I N V I T E D R E V I E W

GFP is the way to glow: bioimaging of the plant endomembrane system

F. B R A N D I Z Z I *, S . L . I R O N S , J. J O H A N S E N , A . KO T Z E R & U. N E U M A N N

Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, U.K.

*

Department of Biology, University of Saskatchewan, Saskatoon, Canada

Summary

It is less than a decade that the green fluorescent protein (GFP)and its spectral variants have changed the approach to studyingthe dynamics of the plant secretory pathway. GFP technologyhas in fact shed new light on secretory events by allowingbioimaging

in vivo

right to the heart of a plant cell. This reviewhighlights exciting discoveries and the most recent develop-ments in the understanding of morphology and dynamics ofthe plant secretory pathway achieved with the application offluorescent proteins.

Received 22 October 2003;

accepted 22 December 2003

1. The plant secretory pathway

The endoplasmic reticulum (ER), the Golgi apparatus, thevacuole and the plasma membrane constitute the major com-ponents of the plant secretory system (Fig. 1). In general, theER appears as the central factory dedicated to the synthesis ofproteins, lipids and glycans. ER proteins that are not retainedin the lumen of the ER or in the ER membrane are processedand folded prior to their transport to the Golgi apparatus(reviewed in Vitale & Denecke, 1999). In the Golgi, proteinsare further processed (reviewed in Dupree & Sherrier, 1998)and then, if not resident in the Golgi, are sorted either to thestorage (for example in pea cotyledons via dense vesicles; Hohl

et al

., 1996) or to the lytic vacuole via prevacuolar compart-ments (PVCs; Matsuoka & Neuhaus, 1999; Vitale & Raikhel,1999) or multivesicular bodies (MVBs; Robinson

et al

., 1998).Alternatively, proteins can be secreted at the plasma membranevia vesicular transport (reviewed in Satiat-Jeunemaitre &Hawes, 1993; Battey

et al

., 1999).Recycling of membranes via an endocytic pathway contrib-

utes to the maintenance of the overall lipid and protein distri-bution between plasma membrane and secretory organelles(reviewed in Hawes

et al

., 1995; in Battey

et al

., 1999; Crooks

et al

., 1999). This would result in a movement of vesicles fromthe plasma membrane to the partially coated reticulum (PCR),and from this to either the Golgi or the MVBs and then to thevacuole (Fowke

et al

., 1991; see also Hawes

et al

., 1999).Our understanding of the dynamics of the secretory path-

way in plant cells is often impaired by the difficulty of workingwith such cells. The dynamics of the secretory pathway havebeen exponentially clarified in recent years, thanks to newtools becoming available, such as green fluorescent protein(GFP) technology, that allow the study of dynamic cellularprocesses

in vivo

. GFP is a self-assembling fluorescent proteinwith a molecular mass of approximately 27 kDa. It emits greenfluorescence upon excitation with UV or blue light. As specificsecretory proteins or signals can be fused to GFP, usuallywithout altering their targeting, it is a useful alternative toconventional dyes previously used to investigate endomem-brane compartments

in vivo

. Moreover, GFP itself is not ableto cross most membranes, with the exception of the nuclearmembrane through the nuclear pores (Grebenok

et al

., 1997).These are some of the most valuable features of GFP and itsspectral derivatives, especially considering that many of thevital dyes available for studies in animal cells cannot be usedfor investigating the biology of plant cells. In this respect,GFP has played a central role in uncovering dynamic endo-membrane events

in vivo

(for a review see also Brandizzi

et al

.,2002b).

1.1.

Introduction of GFP into the plant endomembrane studies

GFP was first identified in the jellyfish

Aequoria victoria

(Shimomura

et al

., 1962) and purified and crystallizedlater by Morise

et al

. (1974). About 20 years later, Prasher

et al

.(1992) cloned the

gfp

gene. In 1994, it was first demonstratedthat the expression of the gene in heterologous organismsgenerates fluorescence upon UV or blue light excitation(Chalfie

et al

., 1994; Inouye & Tsuji, 1994).In plants, the first reports of successful expression of un-

modified wild-type

gfp

using cytoplasmic RNA viruses such

Correspondence to: Dr Federica Brandizzi. Fax: +1 306 966 4461; e-mail:

[email protected]

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as potato virus X and tobacco mosaic virus date back to 1995(Baulcombe

et al

., 1995; Heinlein

et al

., 1995).Owing to the presence of a cryptic intron recognizable by

plant mRNA splicing machinery, GFP technology had a difficultbirth in plant cell studies. Alteration of the codon usage to be

closer to the plant consensus and removal of the cryptic plantintron (Haseloff

et al

., 1997) allowed a wider exploitation ofGFP as a fluorescent reporter for plant cell bioimaging.

2. Plant endomembranes highlighted by GFP

The introduction of GFP into the plant secretory pathwaymirrors a natural pathway of a secretory protein. GFP has infact first illuminated the ER and nuclear envelope (Boevink

et al

., 1996) and then it has made its way to organelles down-stream of the secretory pathway. In this section we highlightthe way that GFP has revealed the secretory pathway andwe focus on recent discoveries linked to GFP targeting andexpression in different secretory organelles.

2.1.

The nuclear envelope

The dynamics of the plant nuclear envelope (NE) are relativelyuncharacterized in comparison with many other membranesof the plant endomembrane system. This is mostly due to alack of

in vivo

markers specific for the plant NE (Meier, 2001;Irons

et al

., 2003). In the search for an NE-specific markerthat works in plants, the N-terminal domain of the humanlamin B receptor (LBR; Ellenberg

et al.

, 1997) was fused toGFP

5

(Haseloff

et al

., 1997). Stable expression of the LBR-GFP

5

fusion protein in tobacco plants and BY-2 suspension cellsgives specific labelling of the NE (Irons

et al

., 2003; Fig. 2a,b).Immobile fluorescent punctate structures were observed ininterphase (Fig. 2a) and dividing cells (arrowhead, Fig. 2c greenimage) and are likely to be membrane stacks arising as a resultof protein over-expression. Similar membrane stacks havebeen observed in yeast cells expressing LBR (Smith & Blobel,1994). During mitosis, the LBR-GFP

5

fluorescence locates totubular membrane structures, co-localizing with a lumenalER marker constructed with the yellow fluorescent protein(YFP) (Irons

et al

., 2003; Fig. 2c), showing that the proteinmoves to the ER membranes during division as in animal cells.

The continuity of the NE and ER ensures that a secretoryprotein, which labels the ER, labels the NE as well. This is illus-trated by GFP targeted to the ER [signal peptide–GFP (sp-GFP);Boevink

et al

., 1996], and of fluorescent proteins targetedto and retained in the ER (e.g. sp-GFP-KDEL, Boevink

et al

.,1996; sp-YFP-HDEL, calnexin-GFP, Irons

et al

., 2003). In thecase of soluble proteins, this is due to the continuity of theNE and ER lumen. For membrane proteins, the nuclear poresallow lateral diffusion of small proteins, with labelling of theinner NE as well as the outer NE and ER. It is therefore interest-ing that in interphase the fluorescence of LBR-GFP

5

localizesexclusively to the NE, suggesting that specific retention signalsretain the protein in the NE.

A GFP fusion of the plant protein, matrix attachment regionbinding filament-like protein (MFP1) associated factor 1 (MAF1;Gindullis

et al.

, 1999) has been located to the NE area in plantcells and shows association with the nuclear matrix and

Fig. 1. Major organelles of the plant endomembrane system. (a)Schematic diagram of the endomembrane organelles of the plantsecretory pathway. Material exiting the ER can be transported to theprotein storage vacuole (1) or passed to the Golgi. At the Golgi, the maintransport pathways are believed to lead to three different destinations: theprotein storage vacuole (PSV) via so-called dense vesicles (2), the plasmamembrane (PM)/apoplast via secretory vesicles (3) or the lytic vacuolevia clathrin-coated vesicles (4). On route to the lytic vacuole or the PM,material passes through the prevacuolar compartment (PVC), whichmay also be the sorting station for material entering from the PM viaendocytosis (5). (b) Transmission electron micrograph of Arabidopsissuspension cultured cells prepared by high-pressure freezing and freezesubstitution, showing different organelles of the plant secretory pathway.CW, cell wall (apoplast); ER, endoplasmic reticulum; G, Golgi apparatus;LV, lytic vacuole; NE, nuclear envelope; PM, plasma membrane; T,tonoplast. Scale bar = 500 nm.

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nuclear periphery as well as structures further from the NE. AGFP fusion of RanGAP, a protein mediating nuclear import viaactivation of Ran GTPases, labels speckle-like structures at theNE and the cytoplasm (Pay

et al

., 2002). Early papers using anMFP1-GFP fusion showed labelling at the nuclear peripheryand through the cytoplasm (Gindullis & Meier, 1999). Thestructures observed at the nuclear periphery in tobaccosuspension cells expressing MFP1-GFP have recently beenshown to be nucleoids of proplastids close to the NE ( Jeong

et al

., 2003). This finding illustrates the need for high-resolutionmicroscopy beyond the light microscopy level for determina-tion of protein location.

A plant protein that exclusively labels the NE remains elusive.

2.2.

ER morphology and dynamics

The first published GFP fusion labelling of the plant ER wasachieved using the potato virus X expression system (Boevink

et al

., 1996). In tobacco leaf cells GFP was targeted to thelumen of the ER by fusing the coding sequence of the signalpeptide of the potato storage protein, patatin, to the N-terminusof wild-type GFP. Substitution of the patatin signal peptidewith the sporamin signal peptide (PVX.sp-GFP) resulted inbrighter fluorescence, presumably reflecting a higher level oftranslocation of GFP into the ER (Boevink

et al

., 1999).It is known that soluble proteins remain in the ER due to a

C-terminal tetrapeptide (K/HDEL) retrieval signal encodedin their primary amino acid sequence (Denecke

et al

., 1992).Proteins escaping the ER and carrying the K/HDEL signal areretrieved by a receptor that recognizes the signal (Semenza

et al

., 1990; Lee

et al

., 1993). The construct PVX.sp-GFP withouta K/HDEL retrieval signal was secreted and accumulated inthe apoplast in older infections. When the KDEL tetrapeptidewas spliced to the carboxyl terminus of GFP, GFP was retainedin the ER (Boevink

et al

., 1996, 1999). With this marker, ERappears as a relatively immobile but locally remodelling poly-gonal tubular network with variously shaped cisternae at thecell cortex, with other more mobile tubules streaming throughthe cytoplasm.

A similar ER pattern has been identified in studies usingGFP spliced to ER native proteins and targeting motifs. Theseinclude the soluble proteins such as sp-GFP-HDEL (Fig. 2d,f )and colour variants (sp-YFP-HDEL; Irons

et al.

, 2003), domainsof calreticulin (Brandizzi

et al

., 2003), BiP (Lee

et al

., 2002)and the membrane protein calnexin (Irons

et al

., 2003), the di-lysine motif GFP-tm-KKXX (Benghezal

et al

., 2000), and acalmodulin-regulated Ca

2+

-ATPase (ACA2p; Hong

et al.

, 1999).ER structures highlighted by GFP are similar to those seen withER probes such as DiOC

6

and hexyl rhodamine B, confirmingthat GFP is correctly targeted to the ER (Quader, 1990).

sp-GFP-HDEL stably expressed in

Arabidopsis

highlightsa characteristic cortical tubular network in leaf cells(Matsushima

et al

., 2002, 2003). In root cells, the ER showedmobile ‘ER bodies’ as well as a fluorescent tubular network(Hawes

et al

., 2001). The ER bodies are 0.5

µ

m wide and 5–10

µ

m in length and are brightly fluorescent. Their presencehas also been demonstrated in wild-type plants by immuno-fluorescence, hence demonstrating that their presence isnot likely to be an artefact of a GFP fusion expression. A form of

β

-glucosidase with an ER retention signal appears to bethe main constituent of the ER bodies (Matsushima

et al

.,2003). The formation of ER bodies in leaves was observedupon wounding (Matsushima

et al

., 2003), suggesting thatER bodies may have a role in wounding response. For instance,

β

-glucosidases may hydrolyse

β

-linked oligosaccharides (e.g.cellobiose) involved in plant defence, hormone activation andcell wall breakdown.

The movement of ER bodies within the ER lumen raisesquestions about how proteins move within the ER. This opensa new area of plant endomembrane study in which fluorescentprotein technology will be a key tool.

As well as providing a wealth of information on the dynamicsof the endomembrane system in ‘normal’ living plant cells,fluorescent probes can visualize morphological changes occurringin plant membranes during a pathogen attack. For example, theeffect of tobacco mosaic virus infection on the ER morphologywas followed with an ER-targeted GFP (Reichel & Beachy,1998; Gillespie

et al

., 2002) and Takemoto

et al

. (2003) have

Fig. 2.

Organelles of the early endomembrane system highlighted by fluorescent fusion proteins. (a) Confocal laser scanning (CLS) micrograph of aninterphase tobacco BY-2 cell expressing LBR-GFP5 (Irons

et al

., 2003). The fusion protein locates to the nuclear envelope. (b) Low-magnification CLSmicrograph of tobacco epidermal leaf cells stably expressing LBR-GFP5. Nuclear envelopes are highlighted by the fusion protein (green image,arrowheads); nuclear contents are labelled with ethidium bromide (merged image, arrows). (c) CLS micrograph of BY-2 cells stably expressing LBR-GFP5(green image) and a soluble ER-marker, spYFP-HDEL (red image; Irons

et al

., 2003), at different stages of division. The upper two cells are at the end ofmitosis, with the NE and phragmoplast fully formed. The lower cell is in anaphase, with the membranes of the mitotic apparatus forming tubularstructures (green image, arrows) as the chromosomes move towards the cell poles. Apart from the punctate structures, the GFP and YFP fusion proteinslabel the same structures in the dividing cell (merged image), suggesting that the LBR-GFP

5

protein locates to the ER during mitosis. (d) CLS micrograph ofa tobacco leaf protoplast transiently expressing sp-GFP-HDEL (Brandizzi

et al

., 2003). The cortical ER highlighted by this construct appears as a loosenetwork of tubules. (e) CLS micrograph of Golgi stacks in a BY-2 cell labelled with GFP-tagged mannosidase I (GmMan1-GFP; Nebenführ

et al.

, 1999). Theimage was created by merging 15 deconvolved images that were taken 0.5

µ

m apart at the centre of a BY-2 cell near the top of the nucleus. Micrographcourtesy of A. Nebenführ. (f ) CLS micrograph of the cortical cytoplasm of a leaf epidermal cell transiently expressing both spGFP-HDEL (green image;Batoko

et al

., 2000) and the Golgi marker ST-YFP (red image; Brandizzi

et al

., 2002a). Golgi stacks are in close proximity to the tubules of the ER network(merged image). N, nucleus. Scale bars: a,d–f = 10

µ

m; b,c = 20

µ

m.

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investigated the effect of oomycete pathogen attack in

Arabidopsis

on ER and Golgi membranes as well as microtubulesand microfilaments labelled with fluorescent proteins.

2.3.

Golgi imaging

in vivo

Studies on the dynamics of the plant Golgi are relatively youngin comparison with their mammalian counterparts, due tothe absence of genuine plant Golgi enzymes to be fused to GFPand used as

in vivo

markers.The first report on the visualization of the Golgi apparatus

in living plant cells with GFP dates back to 1998 (Boevink

et al

., 1998), whereas the mammalian counterpart dates backto 2 years earlier (Cole

et al

., 1996).The Golgi apparatus in living plant cells has been first visu-

alized with two different fluorescent constructs (Boevink

et al

.,1998). GFP was fused (1) to the putative

Arabidopsis

K/HDELreceptor, ERD2, homologous to a yeast and mammalian proteinthat recycles soluble proteins with a K/HDEL tetrapeptideback to the ER, and (2) to the transmembrane domain (TMD)of a rat sialyl-transferase, a mammalian Golgi glycosylationenzyme (ST-GFP). Both GFP-chimeras were found to localizeto fluorescent punctuate mobile structures, revealed to beGolgi stacks by immunogold electron microscopy with a GFP-antibody (Boevink

et al

., 1998). As native sialyl-transferaseshave not been reported in plants, the targeting of the ST-GFPto the plant Golgi suggests a common mechanism in plantsand mammals for targeting and retention of transferases inGolgi membranes.

Subsequently, identification of novel probes has producedmore reports on the dynamics of the Golgi apparatus. Forexample, Nebenführ

et al

. (1999) reported on Golgi targetingin BY-2 cells of a soybean

α

-1,2 mannosidase I, the first enzymeinvolved in the N-linked oligosaccharide pathway, fused toGFP (Fig. 2e). The N-terminal TMD, including the flankingamino acids from a plant N-acetylglucosaminyltransferaseI, has also been fused to GFP and was found to be targetedto the Golgi in

Nicotiana benthamiana

plants with a tobaccomosaic virus-mediated expression (Essl

et al

., 1999). The samegroup reported later on Golgi targeting by an

Arabidopsis

β

-1,2-xylosyltransferase, a glycosyltransferase that is uniqueto plants and some invertebrates (Dirnberger

et al

., 2002).An N-glycan GFP-tagged xylosyltransferase has been foundassociated with Golgi stacks of BY-2 cells preferentially locatedin medial cisternae (Follet-Gueye

et al

., 2003). Dupree’s groupreported on the expression of a GONST1-YFP fusion thatlocalizes to small punctate structures in onion cells, interpretedas being the Golgi apparatus (Baldwin

et al

., 2001).The organization of the plant Golgi apparatus, at light micro-

scope resolution, appears rather different from the mamma-lian Golgi. The plant Golgi is scattered in the cell as smallstacks in the cortical cytoplasm and within

trans

-vacuolarstrands of cytoplasm (Boevink

et al

., 1998; Wee

et al

., 1998;Essl

et al

., 1999; Nebenführ

et al

., 1999).

In leaves, individual stacks of the Golgi apparatus appearclosely associated with the cortical ER network (Boevink

et al

.,1998; Saint-Jore

et al

., 2002; Fig. 2f ). This is particularlyevident when imaging the ERD2-GFP fusion. In mammaliancells, the homologue of the ERD2 protein has been identifiedon the intermediate compartment between the ER and theGolgi and on the Golgi itself (Griffiths

et al

., 1994). In plantcells, the location of the receptor was, until recently, unknown.Confocal microscopy of ERD2-GFP in

Nicotiana

leaves showedthe chimeric protein to be located to Golgi bodies and theER network. This close association of ER and Golgi has led tothe hypothesis, suggested by a few reports based on electronmicroscopy, that the two organelles may have connectionsand function as a secretory unit ( Juniper

et al

., 1982; Harris& Oparka, 1983; Brandizzi

et al

., 2002c). However, thenature and persistence of these connections have yet to beestablished.

Movement of the Golgi over the ER is actin-dependent.Rhodamine–phalloidin staining of tobacco leaf epidermal cellsexpressing ERD2-GFP has revealed a close juxtaposition of theER and the Golgi to the actin cytoskeleton (Boevink

et al

.,1998). More recently, the dynamic association of the Golgiwith actin has been shown in tobacco epidermal cells express-ing fluorescent constructs for Golgi and actin cytoskeleton,the latter being highlighted with an actin-binding region of amouse talin fused to YFP (Brandizzi

et al

., 2002c). Depolymer-ization of the actin network with drugs results in an inhibitionof Golgi movement and clustering of the fluorescent bodieson small islands of lamellar ER within the cortical tubularnetwork (Boevink

et al

., 1998; Brandizzi

et al

., 2002c).The dynamics of the plant Golgi over the ER network opens

up questions on the modality of ER-to-Golgi protein andmembrane transport and on the distribution of ER export sites.It was postulated that ER-to-Golgi protein transport mightbe linked to the discontinuity of Golgi movement (Nebenführ

et al

., 1999). As Golgi stacks move, arrest and regain movementafter time intervals, cargo collection would occur duringGolgi arrest, possibly after transient detachment from actin(‘stop-and-go model’). Alternatively, the Golgi movement inplant cells would allow the Golgi continually to collect vesiclesbudding from the ER (‘vacuum cleaner model’; Boevink

et al

.,1998).

These models, which are as yet unsupported by any exp-erimental evidence, do not exclude the possibility that the ERand Golgi may behave as one dynamic system, either throughdirect membrane continuities or through continuous vesicleor tubule formation/fusion reactions (Brandizzi

et al

., 2002b;reviewed in Neumann

et al

., 2003). In this view, ER-to-Golgitransport may occur during Golgi movement.

2.4. Imaging the vacuole with GFP

The plant vacuole is a multifunctional organelle, which isessential for growth and development. Unlike yeast vacuoles

G F P I S T H E WAY TO G L OW 143

© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158

or mammalian lysosomes, the plant vacuole often serves asboth storage and lytic compartment. In addition, the plantvacuole acts as a pool for metabolic intermediates suchas organic acids and is involved in the regulation of turgorpressure, detoxification of the cytosol and the maintenanceof cytosolic pH.

Both lytic and storage vacuoles may coexist in the same cell(Paris et al., 1997). Lytic and protein storage vacuoles can bedistinguished by the presence of different specific aquaporins,termed tonoplast intrinsic proteins (TIPs). The locationof several TIPs has been established through immunocyto-chemistry. α-TIP has been mainly assigned to protein storagevacuoles ( Johnson et al., 1989). γ-TIP has been found in lyticor degradative vacuoles (Paris et al., 1996; Reisen et al., 2003),whereas δ-TIP has been found in pigment-containing vacuoles( Jauh et al., 1998). The discovery of GFP and the formation ofTIP-GFP fusion proteins has allowed in vivo study of vacuolarfunctions and dynamics.

In a random GFP::cDNA fusion screen, a δ-TIP fused toGFP was demonstrated to be a vacuolar membrane protein inArabidopsis hypocotyl epidermal cells (Cutler et al., 2000). Theidentity of this vacuolar membrane is at present unknown.

Reisen et al. (2003) recently reported on another aquaporinisolated from cauliflower, BobTIP26-1, that has previouslybeen demonstrated to be an active aquaporin in Xenopus leavisoocytes. BobTIP26-1 is a protein located in the tonoplast(Barrieu et al., 1998) and is a specific marker for acidic, lyticvacuoles. Reisen et al. (2003) fused the GFP sequence down-stream of the BobTIP26-1 coding region and observed acomplex tonoplast labelling in Nicotiana tabacum cv. Wisconsin38 suspension cells. Fluorescent patches were detected on thetonoplast, which might suggest that the aquaporins are notevenly distributed within the vacuolar membrane. In tobaccoleaf epidermal cells, BobTIP26-1-GFP evenly labels thetonoplast (Fig. 3a). When BobTIP26-1-GFP was transientlyexpressed in protoplasts isolated from tobacco suspensioncultures, the fusion protein was not detected on the tonoplast,but in a fluorescent network resembling the ER. Reisen et al.(2003) suggest that this might be due to BobTIP26-1-GFP enroute to the vacuolar membrane.

A study using γ-TIP-GFP to investigate the dynamics of thevacuole reported that the tonoplast protein was targeted tothe vacuolar membranes in young Arabidopsis cotyledon cells(Saito et al., 2002). In addition, it was found that the markerprotein also labelled some spherical structures (bulbs) thatwere often observed within the lumen of vacuoles. These bulbswere connected with the vacuolar membrane and weremoving around within or along the outline of the membrane.In addition, larger bulbs were also shown to become elongatedand form tube-like structures. The intensity of the fluorescenceemitted from the γ-TIP-GFP-labelled bulbs was reported to beseveral-fold higher than the adjacent vacuolar membrane.This could be explained by the observation using transmissionelectron microscopy that the bulbs consist of a double

Fig. 3. Fluorescent protein fusions highlights vacuole and plasmamembrane. (a) Low-magnification CLS micrograph of tobacco leafepidermal cells transiently expressing a GFP fusion to the tonoplastintrinsic protein BobTIP26-1 (Reisen et al., 2003). The fusion proteinlocates to the tonoplast of central lytic vacuole, thereby visualizingthe transvacuolar strands (arrowheads). The insert shows cytoplasmicstrands leading to the centre of a cell where the nucleus is located in acytoplasmic pocket in the middle of the vacuole. BobTIP26-1-GFPconstruct courtesy of N. Leborgne-Castel. (b) CLS micrograph of a tobaccoleaf epidermal cell transiently expressing NtAQP1-YFP (A. Kotzer andC. Hawes, unpublished data). This YFP fusion to the plasma membraneintrinsic protein NtAQP1 exclusively labels the plasma membrane,thereby outlining the cell shape. Note that in comparison with (a), nocytoplasmic strands are highlighted. NtAQP1 construct (Siefritz et al.,2001) courtesy of F. Siefritz. (c,d) Projection of CLS optical sectionsthrough the roots of Arabidopsis cv. Wassylevskaja plantlets (Flückigeret al., 2003), at an early stage of root hair formation, stably expressingtwo different vacuolar GFP markers. (c) GFP-Chi (Di Sansebastiano et al.,1998, 2001), a marker of the protein storage vacuole, accumulatesin small compartments. (d) Aleu-GFP (Di Sansebastiano et al., 2001), amarker of the lytic vacuole, accumulates in the large central vacuole.Micrographs courtesy of G. P. Di Sansebastiano. N, nucleus. Scale bars:a–d = 25 µm; insert in a = 10 µm.

144 F. B R A N D I Z Z I E T A L .


membrane. In the same study, another vacuolar membraneprotein, AtRab75c, was fused to GFP and expressed in Arabi-dopsis. The construct gave good vacuolar localization but,interestingly, did not label any structures in the lumen of thevacuole as seen for the γ-TIP-GFP (Saito et al., 2002). However,although not fluorescent, bulbs are present in cotyledonepidermal cells in GFP-AtRab75c-transformed Arabidopsisas confirmed by electron microscopy. γ-TIP is an integralmembrane protein with six TMDs and probably forms a dimeror tetramer like other aquaporins, whereas Rab75c belongs tothe Rab GTPase family and is attached to the membrane viaa C-terminal prenylation. Saito et al. (2002) proposed that thedifference in membrane attachment might be the reason fortheir segregation into different structures. Alternatively, anunknown mechanism might exclude GFP-AtRab75c fromthe bulb region (Saito et al., 2002).

Uemura et al. (2002) used a vacuolar syntaxin-relatedmolecule, AtVam3/SYP22, fused to GFP, to study the vacuolardynamics in Arabidopsis. This protein has previously beendemonstrated by immunoelectron microscopy to locate tothe vacuolar membrane (Sato et al., 1997). The GFP constructrevealed complicated vacuolar architectures in various celltypes with internal membranous structures inside the centralvacuole in root epidermal cells. This was also observed inmesophyll cell protoplasts. By three-dimensional modellingand reconstruction from confocal images, the internalmembrane structures were organized as sheets and cylindri-cal structures. These structures were also highly dynamic andwere constantly remodelled. Interestingly, the fluorescenceintensity of this GFP construct, GFP-Vam3, on the cylindricalstructures was twice as strong as the outer-vacuolar membrane(Saito et al., 2002). Uemura et al. (2002) concluded that thisintravacuolar membrane has a double membrane structure.The streaming motion could be blocked with an actin-depolymerizing agent, cytochalasin D, whereas microtubule-disrupting drugs had no effect on the movement of the sheet-likestructures. These data suggest that the dynamic movementof the internal vacuolar membrane is actin-dependent, butindependent of microtubules. Uemura et al. (2002) concludedthat these sheet-like structures are possible transvacuolarstrands involved in cytoplasmic streaming.

They postulated two models for the formation of intravacu-olar structures. In the ‘autophagic’ model, a small vacuole isengulfed by a large vacuole, and remains enclosed in thevacuolar lumen. The second model suggests that the vacuolarmembrane could be invaginated into the vacuolar lumen toform sheet-like structures, which then become rounded.

A potassium channel protein (AtKCO1) fused to GFP locatesto the vacuolar membrane in transgenic tobacco BY-2 cells(Czempinski et al., 2002). Confocal images showed that thesignal of the AtKCO1-GFP was present as a single fluorescentline surrounding the large central vacuole. In addition,individual cells contained multiple small vacuoles labelledby GFP fluorescence. In addition, in some cells containing a single

large vacuole, a network of connecting strands most similar totransvacuolar strands exhibited GFP fluorescence. Likewise,an iron-regulated ABC transporter, IDI7, from barley roots fusedto GFP localized to the vacuolar membrane when transientlyexpressed in suspension-cultured tobacco cells (Yamaguchiet al., 2002). IDI7 is believed to be a tonoplast ABC transporterand is thought to be involved in the export of certain sub-strates from the cytosol to the vacuoles. Finally, Thomine et al.(2003) expressed a metal transporter of the NRAMP family,AtNRAMP3, as a GFP fusion in onion cells and in Arabidopsisprotoplasts located to the vacuolar membrane.

The existence of more than one type of vacuole in some cellsimplies that, in these cases, different mechanisms for tono-plast protein sorting coexist. It has been established for severalvacuolar membrane proteins that both the TMD and thecytosolic tail are important for sorting to the tonoplast ( Jiang& Rogers, 1998). A C-terminal portion of α-TIP composed ofthe sixth and last TMDs and the 17-amino-acid cytosolic tailcontains sufficient information for sorting to the tonoplast(Hofte & Chrispeels, 1992).

Unlike the increasing amount of data collected using GFPas a tag for soluble vacuolar proteins, little has been done toinvestigate the actual transport pathway of vacuolar mem-brane proteins using GFP. Mitsuhashi et al. (2000) designed α-TIP-GFP and γ-TIP-GFP constructs and both were localized toa vacuolar membrane in BY-2 cells. Mitsuhashi et al. (2000)concluded that both TIP-GFP fusions are targeted to the samevacuole in BY-2 cells. This is in contrast to what Okita & Rogers(1996) and Paris et al. (1996) reported, in which the two TIPslocalize to different vacuoles in the same cell in barley rootsand in maturing pea cotyledons. It will be interesting to rein-force these findings on the dynamics of coexisting vacuoleswith experiments based on co-expression of different TIPchimeras with available GFP spectral variants. In the experi-ments of Mitsuhashi et al. (2000), GFP fluorescence was alsoobserved in the ER network, suggesting that both α-TIP-GFPand γ-TIP-GFP are transported through the ER and then to thevacuolar membranes.

2.5. Plasma membrane illuminated by GFP

The plasma membrane (PM) encloses the cell and is the primebarrier between the cytosol and the extracellular environment.The PM is a dynamic, fluid structure constantly changingin composition and organization. Fusions of various plasmamembrane proteins to GFP have been used to label the PM, butto date GFP has shed little light on the underlying mechanicsand dynamics of the PM.

GFP fusion proteins targeted to the PM have been con-structed including artificial markers BP22-GFP and TM23-GFP (Brandizzi et al., 2002a). BP22-GFP was designed byadding three hydrophobic residues (LAL) to the TMD of thepea vacuolar sorting receptor BP80 (Paris et al., 1997). Thisaddition resulted in fluorescence accumulation on the plasma



membrane and in small punctate structures (Brandizzi et al.,2002a). Like BP22-GFP, TM23-GFP, a fusion of the TMD of ahuman lysosomal protein (LAMP1) to GFP, was targeted to thePM (Brandizzi et al., 2002a).

In addition, the location of several endogenous/nativeplasma membrane proteins has been demonstrated by theuse of GFP. PM proteins fused to GFP include the Rac-typesmall GTPase ZeRAC2 (Nakanomyo et al., 2002), the tobaccoPM aquaporin NtAQP1 (Siefritz et al., 2001; A. Kotzer andC. Hawes, unpublished observations; Fig. 3b), the ammoniumtransporter LjAMT2;1 (Simon-Rosin et al., 2003) and the acyl-CoA binding protein ACBP1 (Li & Chye, 2003).

2.6. GFP and the apoplast

A GFP construct with the ER signal peptide but lacking theretrieval signal (sp-GFP) has proven to be a useful marker forfollowing secretion in vivo (Boevink et al., 1999). Tobacco leavesinfected with this construct show very little fluorescence inthe ER, probably due to continuous secretion. Treating thesame leaves with cold shock or brefeldin A (BFA) caused aninhibition of the GFP secretion, resulting in the build-up offluorescence in the ER (Boevink et al., 1999). These results weresimilar to those reported for mammalian cell systems (Presleyet al., 1997). As will be discussed in detail below, transport of asecreted form of GFP to the apoplast can also be inhibited byco-expression of dominant-negative mutants of proteinsregulating secretion.

3. GFP sheds light on protein retention and targeting to the endoplasmic reticulum

Mechanisms by which the cell exports secretory proteins butretains resident ER proteins are the subject of intense investi-gation. Brandizzi et al. (2002a) investigated the influence ofthe TMD length on the destination of membrane proteinsalong the secretory pathway. The length of the TMD has beenfound to be important for protein progression along the secre-tory pathway. In this respect, a GFP fusion to a TMD stretchof 17 amino acids (aa) has been located to the NE and ER intobacco. Extension of this stretch by three further aa resultedin the location to the Golgi, and a further elongation of theTMD by another three aa resulted in the GFP fusion incorpora-tion into the PM. This study highlights an analogous influenceof the TMD length on the destination of membrane proteins inplants as found in yeast (Munro, 1995).

Irons et al. (2003) reported a TMD and cytosolic tail GFPfusion of an Arabidopsis calnexin, which contains a dilysinemotif. The chimera locates to the NE and ER and it is highlymobile in these membranes as revealed by fluorescencerecovery after photobleaching (FRAP) technique (Fig. 4a).This construct may be retained in the ER by virtue of specificsignals present in the cytosolic tail of calnexin. Benghezal et al.(2000) have shown that the C-terminal dilysine motif confersER localization to type I membrane proteins in plants. Cf-9 (aresistance gene, conferring resistance against the fungal path-ogen Cladosporium fulvum) is a type I membrane protein that

Fig. 4. Protein movement into organelles of the endomembrane system as shown by FRAP. (a) Photobleaching of a nuclear envelope (arrow) of a Nicotianatabacum leaf epidermal cell transformed with a calnexin-GFP construct (Irons et al., 2003). The images are prebleach (0.0 s), bleach event (7.4 s), partialrecovery (13.3 s) and full recovery (34.9 s). The frame indicates the area of photobleaching. (b) Photobleaching of a Golgi body in an N. tabacum leafepidermal cell expressing ERD2-GFP and treated with cytochalasin D as described in Brandizzi et al. (2002c). The images are prebleach (0.0 s), bleachevent (7.1 s), partial recovery (141.5 s) and full recovery (300.8 s). The frame indicates the area of photobleaching. Note that the recovery of fluorescencein the nuclear envelope is much faster than the in the Golgi apparatus. Scale bars: a = 10 µm; b = 5 µm.



has a large extracytosolic leucine repeat domain, a single TMDand a cytosolic tail carrying a C-terminal dilysine KKXX motif.To study the ER retrieval mechanism motif of Cf-9, fusions ofGFP to the TMD and cytosolic tail of Cf-9, creating GFP-KKXX,were generated (Benghezal et al., 2000). The location of GFP-KKXX in the ER was verified by the co-localization of BiP andGFP using double immunofluorescent labelling. Amino acidsubstitution of KKXX to NNKK caused the secretion of mostof the fusion protein from the ER, proteolytic release of GFPfrom its membrane anchor and loss of fluorescent properties.The extent to which the GFP-NNKK fusion was retained inthe ER differed between different systems. In epidermal cells,the GFP-NNKK still showed an ER pattern but the intensity offluorescence was much weaker than GFP-KKXX-expressingcells. In BY-2 cells, this was not the case as the GFP-NNKKshowed no ER fluorescence.

Soluble reticuloplasmins are targeted to the ER by virtue of asignal peptide. Signal peptides, like those of sporamin, chitinaseand patatin, were proven sufficient for targeting GFP to theER lumen (Boevink et al., 1996; Batoko et al., 2000; Brandizziet al., 2003).

The ER retention of soluble reticuloplasmins in plantsdepends on the carboxyl-terminal signal K/HDEL (Deneckeet al., 1992). As mentioned above, ER-targeted GFP fused to K/HDEL is retained in the ER (Boevink et al., 1996; Brandizzi et al.,2003). However, when overexpressed, ER-targeted GFP-HDEL(sp-GFP-HDEL) may reach the vacuole (Brandizzi et al., 2003).This is consistent with the retrieval mechanism of reticulo-plasmins being under the control of a saturable pathway. Atransmembrane receptor ERD2 has been identified in Arabidopsis(Lee et al., 1993) and is likely to function in the same way asthe yeast and mammalian homologues in recognizing the ERretention motif and to trigger retrograde transport of solubleER-resident proteins that have escaped. The localization of ERD2-GFP on the Golgi and ER reflects the fact that the receptor islikely to shuttle between the two organelles. However, the activ-ity of the ERD2-GFP fusion has not yet been tested in plants.

Malfolded secretory proteins are retained in the ER untilthey reach a final low-energy state conformation. How reten-tion is accomplished is still a matter of debate. It is temptingto suggest that retention may be operated by lumenal chaper-ones or alternatively that malfolding may prevent proteinsfrom being recognized by the ER-export machinery. If proteinsdo not reach a proper conformation, they are generallydegraded. In plants, little is known about the mechanisms andproteolytic systems that mediate protein quality control in thesecretory system. It is known that malfolded lumenal proteinscan be targeted to the vacuole via a Golgi-mediated pathway(Pueyo et al., 1995; Coleman et al., 1996). Other malfoldedproteins are degraded via a BFA and heat-shock-independentpathway in an unidentified cellular compartment (Pedrazziniet al., 1997). The existence of a protein retrograde transportand degradation pathway in plant cells has been suggestedby the analysis of the location of the ricin catalytic A subunit

in tobacco protoplasts (Frigerio et al., 1998). Brandizzi et al.(2003) have identified a GFP fusion that is detected in thecytosol and the nucleoplasm of tobacco cells in spite of thepresence of an N-terminal secretory signal peptide. In contrastto secreted GFP, the fusion protein is retained in the cells whereit is degraded slowly, at a higher rate than the ER retainedGFP-HDEL. The fusion protein could not be stabilized by inhib-itors of transport or the cytosolic proteasome. Brandizzi et al.(2003) suggested that the fusion protein is disposed of fromthe ER via a retrograde translocation back to the cytosol.Moreover, accumulation in the nucleoplasm was shown to bemicrotubule-dependent, unlike the well-documented diffusioninto the nucleoplasm of cytosolically expressed GFP. The appar-ent active transport of the GFP fusion into the nucleoplasmmay indicate a yet undiscovered feature of the ER-associatedprotein degradation pathway and may explain the insensitivityof degradation to proteasome inhibitors (Brandizzi et al., 2003).

4. GFP highlights protein movement towards cell compartments

4.1. GFP shows that ER-to-Golgi transport does not require cytoskeleton

GFP is an important tool for investigating ER-to-Golgi proteintransport in vivo. Saint-Jore et al. (2002) used BFA to establishthe involvement of the cytoskeleton in the transport to and fromthe Golgi. In mammalian cells, BFA induces redistributionof Golgi membrane proteins to the ER (Lippincott-Schwartzet al., 1990; Robineau et al., 2000). After treatment with BFA,fluorescent Golgi markers expressed in tobacco epidermal leafcells and in BY-2 cells are redistributed to the ER (Boevink et al.,1998; Ritzenthaler et al., 2002; Saint-Jore et al., 2002). TheBFA effect is reversible upon drug washout (Satiat-Jeunemaitre& Hawes, 1993; Satiat-Jeunemaitre et al., 1996; Saint-Joreet al., 2002). Saint-Jore et al. (2002) proved that in the absenceof an intact actin and microtubule cytoskeleton, the BFA effectand recovery were still taking place, indicating that movementof proteins to and from the Golgi is cytoskeleton-independent.

These data were confirmed by Brandizzi et al. (2002c), whoused FRAP (Fig. 4b) experiments to investigate the require-ments of an intact cytoskeleton for fluorescence recoveryinto Golgi. FRAP experiments were performed on leaf tissuesexpressing either ERD2-GFP or ST-GFP, and treated with cyto-skeleton inhibitors. When the FRAP protocol was appliedto cells treated with actin depolymerizing agents, in order tostop Golgi movement, the recovery of fluorescence in bleachedGolgi stacks occurred within 5 min of the bleaching event(Fig. 4b). This shows that actin is not required for ER-to-Golgitrafficking even though it is required for Golgi movement. AsGolgi still moved in the absence of microtubules, the concomi-tant use of an actin depolymerizing agent was required toperform FRAP on immobile Golgi. Again, Golgi bodies re-gainedfluorescence within 5 min after the bleaching, indicating that



neither actin filaments nor microtubules are necessary forER-to-Golgi protein transport. These results highlight a majordifference between the plant and mammalian Golgi, as itappears that in plants, the cytoskeleton is not essential forprotein transport to the Golgi. Moreover, although the Golgimarker ERD2-GFP was found distributed uniformly over theGolgi and ST-GFP towards the trans-most cisternae (Boevinket al., 1998), no substantial differences in the recovery timesupon photobleaching were recorded. In addition, the recoverytime of these two proteins was similar to the recovery time of aglycosylated form of ST-GFP (Batoko et al., 2000).

4.1.1. Out of the Golgi: transport to the vacuole. A considerableeffort has been made over the last few years to identify andcharacterize differentiated and specialized vacuoles in higherplants. Recently, GFP has become a popular tool to unravel themechanisms of transport of soluble and membrane proteinsto the different vacuoles and to investigate the dynamics of theplant vacuolar system. However, the acidic nature of certaintypes of vacuoles has made it difficult to explore the accumula-tion of certain soluble markers, such as aleurain-GFP, for therapid degradation/quenching of GFP (Di Sansebastiano et al.,1998). The low pH may be a determinant feature of vacuoleswith a primarily lytic function, and storage vacuoles need notbe acidic. Recently, using Arabidopsis cv. Columbia seedlings,it has been shown that light-dependent hydrolytic enzymesinfluence the stability of GFP and account for the reductionof GFP fluorescence in lytic vacuoles (Tamura et al., 2003).However, Di Sansebastiano et al. (2004) have not shown asimilar light-dependent effect on fluorescence levels of GFPin lytic vacuoles when using another cultivar (Arabidopsis cv.Wassylevskaja), suggesting that different ecotypes within thesame species may account for differences in the visibility ofGFP in the vacuolar system (Di Sansebastiano et al., 2004).

Soluble proteins reach the vacuole via information storedin their C-terminus, N-terminus or in an internal fragment oftheir propeptides. In the absence of such signalling peptides,vacuolar proteins are secreted. Cleavable vacuolar sortingsignals include the N-terminal propeptide (NTPP) present insweet potato sporamin and the C-terminal propeptide (CTPP)present in barley lectin (reviewed in Matsuoka & Neuhaus,1999; Vitale & Raikhel, 1999). The N-terminal NTPP signalscontain an NPIR consensus amino acid motif that is necessaryfor targeting sporamin to the vacuole (Matsuoka et al., 1995).A consensus sequence has not yet been identified in theC-terminus, but rather a common structural motif seems toserve as a sorting signal in the CTPPs (reviewed in Matsuoka& Neuhaus, 1999). CTPP- and NTPP-dependent pathwaysare likely to be distinct.

Soluble proteins destined for the lytic vacuoles, such asbarley aleurain, are transported through the Golgi complex.From the Golgi apparatus, the proteins are transported to aprevacuolar compartment (PVC) in clathrin-coated vesicles(CCVs; Paris et al., 1996; Vitale & Raikhel, 1999). A type I trans-

membrane glycoprotein, BP80, probably acts as a receptor forprotein delivery to the lytic vacuole (Matsuoka et al., 1997;Paris et al., 1997).

Lumenal vacuolar proteins might reach the protein storagevacuole in three different ways. The first route is throughautophagy of ER-released storage protein aggregates (Levanonyet al., 1992). In the second route, precursor-accumulating(PAC) vesicles may be released from the ER and mediatetransport of storage proteins directly to the storage vacuole asreported in pumpkin and castor bean seeds (Hara-Nishimuraet al., 1998). The third route includes transport via the Golgicomplex where storage vacuole proteins are sorted in densevesicles from other proteins in the secretory pathway asdemonstrated for barley lectin (Hohl et al., 1996). Each trans-port pathway implies a first sorting event in the ER wheresome proteins are transported to the Golgi complex whereasothers would aggregate and be released in the cytosol or bepacked into PAC vesicles for transport to the storage vacuole.

Di Sansebastiano et al. (2001) described protein traffickingto two different types of vacuoles in protoplasts by the fusionof GFP to two vacuolar sorting determinants (VSD). One GFP,mGFP5, was targeted to a pH-neutral vacuole by the C-terminalvacuolar sorting signal (VSS) of tobacco chitinase A (see alsoFig. 3c). The construct was demonstrated to accumulate in anorganelle different from the organelle accumulating the stainneutral red, which on proteonation is trapped within acidiccompartments. It was concluded that the fusion to the C-terminus VSS of chitinase A is sufficient to send a secreted GFPto the storage vacuole. A construct made by fusing the NTPPof barley aleurain to mGFP5 was localized to compartmentssmaller than 2 µm and no fluorescence was detectable in themain lytic vacuole. The authors suggested that the lack offluorescence in the main lytic vacuole might be due to therapid degradation of GFP in acidic environments and thatthe observed fluorescent smaller compartments might beintermediates in the transport pathway of aleurain (see alsoFig. 3d). Fusion of the pro-peptide of aleurain to a brighter andmore stable form of GFP, mGFP6, targeted the construct to thesmall intermediate compartments in addition to the centralvacuole. Neutral red was demonstrated to accumulate inthe same compartment as mGFP6-aleurain, indicating a lyticvacuolar location for aleurain. This also clearly indicates thesensitivity of mGFP5 to an acidic environment.

Recently, Flückiger et al. (2003) made stable Arabidopsistransformants with soluble vacuolar GFP constructs. It wasevident that the distribution of the fluorescence of GFP-Chiand GFP-Aleu was different in elongated root cells. The GFP-Chi was located in the ER, small vacuoles and occasionally inthe large central vacuole, whereas GFP-Aleu accumulated inthe large lytic vacuole (Fig. 3c,d). In addition, the organizationof the vacuolar system was investigated in different tissues ofArabidopsis cv. Wassylevskaja plantlets. In contrast to thelocation of GFP-Chi in elongated root cells, GFP-Chi was foundin root apex cells to accumulate in the ER and in different



smaller vacuolar compartments. These results highlight thedifferences in protein targeting due to tissue specificity andcellular differentiation (Flückiger et al., 2003).

The location of another soluble vacuolar protein, 2Salbumin, a major storage protein, was investigated using GFP(Mitsuhashi et al., 2000). A construct, consisting of a signalpeptide (sp) fused to GFP followed by the C-terminal 18-aapeptide (2SC), sp-GFP-2SC, was found to be able to bind toPV72, a putative sorting receptor for the storage vacuole.Fluorescence was observed in a large compartment as well asin the ER network and in the nuclear envelope. The large com-partment was also stainable with BCECF, which is fluorescentin vegetative vacuoles in BY-2 cells. Some small particles werealso labelled by sp-GFP-2SC. Mitsuhashi et al. (2000) postu-lated that because these particles show a similar size as theGolgi complex, the C-terminal peptide of 2S albumin functionsas a targeting signal for vacuoles via the Golgi complex.

Mitsuhashi et al. (2000) also designed a GFP fusion of sp-GFPand the C-terminal region of pumpkin PV72 that includedthe TMD and the cytosolic tail. This construct located to smallparticles in 3-day-old BY-2 cells. The authors concluded thatthese particles were consistent with fluorescent Golgi com-plexes and that the C-terminal part of PV72 is responsible forthe presence of the receptor in the Golgi complex. An observa-tion on these experiments is that no co-localization with aGolgi marker was performed, and that these ‘small particles’might also correspond to PAC vesicles that Hara-Nishimuraet al. (1998) previously demonstrated PV72 to locate to. In10-day-old callus cells, the fluorescence of sp-GFP-PV72 wasobserved within the vacuoles and the construct was demon-strated to be proteolytically cleaved by immunoblot analysis.Mitsuhashi et al. (2000) suggest that like pea BP-80, thevacuolar sorting receptor for the lytic vacuole, PV72, mightcycle between the Golgi complex and a prevacuolar compart-ment of the storage vacuole.

5. GFP technology helps to locate regulatory small GTPases and to shed light on their function

In eukaryotic cells, the model that transport vesicles mediateprotein transport between the various compartments of theendomembrane system is generally accepted. The formation,transport and fusion with a target membrane of these vesicularshuttles are orchestrated by a plethora of proteins. Amongthese regulatory proteins, Ras-related small GTPases playan important role (Vernoud et al., 2003). Whereas Arf andSar GTPases are predominantly involved in the formation ofvesicles and cargo packaging (Chavrier & Goud, 1999), Rabproteins are mainly thought to act as key regulators of thefusion of vesicles with their appropriate target membrane(Zerial & McBride, 2001).

GFP technology has increased our understanding of thefunction of small GTPases in trafficking events betweencompartments of the plant endomembrane system (Figs 5–8).

Two major GFP-based approaches have been adopted. Thefirst consists of expressing GFP fusions of small GTPases inorder to identify their subcellular distribution, which can beindicative of the organelles involved in the transport step(s)regulated by that particular gene product (Ueda et al., 2001;Cheung et al., 2002; Inaba et al., 2002; Bolte et al., 2004). Thesecond approach is based on different GFP markers either ofone specific endomembrane organelle or of a whole transportpathway (Andreeva et al., 2000; Batoko et al., 2000; Saint-Joreet al., 2002; Sohn et al., 2003). In co-expression experiments,the influence of small GTPases on the intracellular distribu-tion/transport of a given GFP marker can then be studied withthe confocal laser scanning microscope. In this type of study,defined point mutations predicted to disrupt GTP binding andhydrolysis activity of small GTPases (resulting in constitutivelyactive or inactive mutant forms) are often introduced in theircoding sequence. The influence of the wild-type and the differ-ent mutant forms on the intracellular distribution/transportof GFP markers can then be compared. Finally, in a combina-tion of both approaches, dual colour imaging experiments areperformed in which fluorescent protein-tagged small GTPases(i.e. GFP) are co-expressed with fluorescent markers based ona spectral derivative [i.e. YFP, and the cyan fluorescent protein

Fig. 5. Effect of wild-type and mutant forms of Sar1p on the transportof Golgi and vacuolar fluorescent markers. (a–c) CLS micrographs ofArabidopsis protoplasts expressing ERD2-GFP, a Golgi/ER marker, alone(a) and alongside AtSar1 (b) or AtSar1 H74L (5c). When expressed alone(a) or with wild-type AtSar1 (b), GFP fluorescence is mainly in the form ofpunctate structures corresponding to individual Golgi stacks, whereasco-expression of AtSar1 H74L leads to fluorescence accumulation ofAtErd2-GFP in an ER-like pattern (c). (d–f) CLS micrographs of Arabidopsisprotoplasts expressing a GFP fusion to the vacuolar soluble proteinsporamin alone (d) and alongside AtSar1 (e) or AtSar1 H74L (f ). Whenexpressed alone (d) or with wild-type AtSar1 (e), sporamin-GFP locates tothe lumen of the central vacuole and to additional punctate structures,whereas when co-expressed with AtSar1 H74L, sporamin-GFP fluorescenceaccumulates in the ER (f ). Scale bars = 10 µm. (a) to (f ) are from Takeuchiet al. (2000) (figs 3a,c,e, 7a,c,e). Copyright Blackwell Publishing, Oxford,U.K., and reprinted with permission.



(CFP)]. This last method offers the possibility to correlateunequivocally, in a cell population, the phenotype of individualcells to the effect of GTPases.

5.1. Regulatory proteins of ER-Golgi/Golgi-ER transport steps

5.1.1. Sar1p. In mammalian and yeast cells, protein transportfrom the ER to the Golgi is mediated by COPII-vesicles. Cargo

packaging and vesicle formation at this level requires initialbinding to the ER membrane of the Sar1p GTPase (reviewed byBarlowe, 2002). Two tobacco and Arabidopsis Sar1p homo-logues were shown to be able to complement the lethal yeast∆sar1 mutant (Takeuchi et al., 1998). Co-expression of one oftwo mutant forms of the tobacco Sar1p impaired in guaninenucleotide interactions (T34N and H74L) with either asecreted soluble or a Golgi membrane GFP marker led to anaccumulation of fluorescence in the ER of Nicotiana clevelandiileaf epidermal cells (Andreeva et al., 2000). Fluorescence alsoaccumulated in the ER, though to a lesser extent, when theGFP markers were co-expressed with a third mutant form oftobacco Sar1p (N129I) and with an entire wild-type ORF inanti-sense orientation (Andreeva et al., 2000). Similar resultswere obtained by Takeuchi et al. (2000), in which one of twoGolgi GFP markers and a wild-type or mutant form (H74L) ofArabidopsis Sar1p (AtSar1) were encoded in the same T-DNA.The Golgi markers relocated from the Golgi to the ER in tobaccoBY-2 cells as well as in Arabidopsis suspension protoplastswhen simultaneously expressed with AtSar1 H74L (Takeuchiet al., 2000; Fig. 5a–c). In addition, AtSar1 H74L, in contrast tothe wild-type protein, had a drastic effect on the intracellulardistribution of a soluble GFP marker targeted to the lytic vacuole(sporamin-GFP). In Arabidopsis suspension protoplasts, fluo-rescence distribution of sporamin-GFP changed from a predom-inantly vacuolar location to an ER-like network pattern(Takeuchi et al., 2000; Fig. 5d–f ). A biochemical approachconfirmed that Sar1p is necessary for transport of proteins tothe Golgi in tobacco leaf protoplasts (Phillipson et al., 2001).

5.1.2. Arf1, Arf3. Arf1 GTPases are involved in vesicle bud-ding steps by recruiting COPI coatomer components as wellas clathrin coats. In mammalian cells, COPI vesicles mediateretrograde protein transport between the Golgi cisternae andfrom ER-Golgi-intermediate compartments back to the ER;their involvement in anterograde protein transport is stilldebated (Spang, 2002). In plant cells, Arf1 has been locatedto the Golgi by immunocytochemical means both by lightmicroscopy in BY-2 cells (Ritzenthaler et al., 2002) and inmaize root tip cells (Couchy et al., 2003), as well as by electronmicroscopy in maize and Arabidopsis root tip cells (Pimpl et al.,2000). In a GFP-based study, Arabidopsis Arf1-GFP (AtArf1-GFP)was shown to accumulate in BFA-sensitive, Golgi-reminiscentpunctate fluorescent structures in both Arabidopsis protoplastsand BY-2 cells (Takeuchi et al., 2002). The effect of differentforms of AtArf1 on various fluorescent markers (three Golgimarkers, one vacuolar marker) was evaluated principallyin tobacco BY-2 cells using an approach similar to the onedescribed above for Sar1p in which the marker protein and theuntagged small GTPase are expressed from the same T-DNA(Takeuchi et al., 2000). Expression of the GTP-locked AtArf1Q71L or the GDP-locked AtArf1 T31N resulted in the reloca-tion of ERD2-GFP fluorescence from the Golgi to the ER, whereastwo other fluorescent Golgi markers were not affected or at

Fig. 6. Effect of wild-type and mutant forms of Arf1 and Arf3 on thetransport of Golgi and plasma membrane GFP markers. GFP fluorescencein green, chlorophyll autofluorescence in red. (a–c) CLS micrographs ofArabidopsis protoplasts transiently expressing the Golgi marker ST-GFPalongside Arf1 (a) or Arf1[T31N] (b) or Arf3[T31N] (c). When expressedwith wild-type Arf1 (a) or Arf3[T31N] (c), GFP fluorescence is in the formof punctate structures corresponding to individual Golgi stacks, whereasco-expression of Arf1[T31N] leads to a diffuse GFP pattern (b). (d–g) CLSmicrographs of Arabidopsis protoplasts transiently expressing the plasmamembrane marker H+-ATPase-GFP alone (d) and alongside Arf1 (e) orArf1[T31N] (f,g). When expressed alone (d) or with wild-type Arf1 (e),H+-ATPase-GFP is exclusively located at the plasma membrane (arrows).Co-expression with Arf1[T31N] leads to fluorescence accumulation ofthe plasma membrane marker in punctate structures and aggregates (f,g,arrowheads). CH, chloroplasts. Scale bars: d–e = 20 µm. (a)–(c) are fromLee et al. (2002) (fig. 2A,a,b,c). (d) to (g) are from Lee et al. (2002)(fig. 6A,a,b,c-3,c-2). Copyright the American Society of Plant Biologistsand reprinted with permission.



least did not relocate to the ER. In addition, the transportof sporamin-GFP to the lytic vacuole was inhibited with themarker accumulating in the ER when AtArf1 Q71L waspresent. The authors suggested that Arf1 is primarily requiredfor retrograde Golgi-to-ER trafficking and that disruption ofArf1 activity leads indirectly to perturbation of ER-to-Golgitrafficking (Takeuchi et al., 2002).

Functional differences between the two Arabidopsis isoformsArf1 and Arf3 were described by Lee et al. (2002) in experimentsperformed in Arabidopsis leaf protoplasts in which either thewild-type or GDP-binding dominant-negative mutants of Arf1and Arf3 (Arf1[T31N] and Arf3[T31N]) were co-transfectedalongside one or two cellular markers tagged by either GFP orthe red fluorescent protein (RFP). In the presence of Arf1[T31N]but not of Arf1, Arf3 and Arf3[T31N], the Golgi markerST-GFP relocated to the ER, which was found to displayprofound morphological changes (Fig. 6a–c). Arf1[T31N] andArf3[T31N] also had a different effect on the traffickingof H+-ATPase-GFP. Although the presence of Arf1 and Arf3wild-type as well as of Arf3[T31N] did not affect the targetingof H+-ATPase:GFP to the plasma membrane (Fig. 6d,e), H+-ATPase:GFP fluorescence was in the form of punctate stainsor aggregates and not at the plasma membrane or in the ERwhen co-expressed with Arf1[T31N] (Fig. 6f,g). Interestingly,BFA caused a similar change in distribution of H+-ATPase:GFP,suggesting that Arf1 may act via a BFA-sensitive factor as inyeast and mammalian cells (Lee et al., 2002).

These reports clearly show that Arf1 mutants do not alwayslead to a relocation into the ER of markers of different cellularcompartments (Golgi, vacuole, plasma membrane) althoughall of these markers are transported from the ER to the Golgi(AtArf1 Q71L even had different effects on markers of the sameorganelle, the Golgi). This indicates that inhibition of transportby Arf1 mutants cannot be easily explained either by a generalblockage of anterograde ER-to-Golgi transport or by a blockageof retrograde Golgi-to-ER transport. In this respect it is inter-esting to note that biochemical data indicate that Arf1 mightnot only influence transport steps of soluble cargo to the plasma

membrane but also have an influence on the BP80-mediatedtransport of soluble cargo to the vacuole (Pimpl et al., 2003).

5.1.3. Rab1 and Rab2 homologues. Whereas Sar1p and Arf1linked to vesicle formation and recruitment of vesicle coatproteins, other small GTPases act predominantly at the level ofvesicle fusion with the target membrane. One example for asmall GTPase apparently involved at this particular transportlevel of anterograde ER-to-Golgi protein trafficking is Rab1,controlling tethering and fusion events at the Golgi level(Lupashin & Waters, 1997; Moyer et al., 2001). Batoko et al.(2000) used both biochemical and GFP techniques to investi-gate the function of an Arabidopsis Rab1 (AtRab1) homologuein tobacco. Co-expression of the dominant inhibitory mutantAtRab1b(N121I) alongside a secreted form of GFP (secGFP)resulted in GFP fluorescence accumulating in tobacco leafepidermal cells in an ER-reminiscent dynamic reticulatepattern, whereas the fluorescence pattern of secGFP was un-altered when co-expressed with wild-type AtRab1b (Fig. 7a–e).AtRab1b(N121I), when co-expressed alongside the Golgimarker ST-GFP, was shown to have no effect on the BFA-induced redistribution of the Golgi marker back into the ER(Saint-Jore et al., 2002). However, recovery of Golgi fluores-cence upon BFA removal was seriously reduced in the presenceof AtRab1b(N121I) (Fig. 7f,g) whereas the BFA recoveryphenotype could be rescued when the wild-type protein wasco-expressed alongside AtRab1b(N121I). These data stronglysuggest that AtRab1b regulates anterograde rather thanretrograde transport between the ER and the Golgi. However,although providing strong evidence for the involvement ofRab1 in ER-to-Golgi trafficking events in plant cells, neither ofthe two studies (Batoko et al., 2000; Saint-Jore et al., 2002)could distinguish between a blockage of protein export outof the ER and blockage of protein import into the Golgi.

Tobacco Rab2 (NtRab2), a homologue of the mammalianRab2 GTPase, has recently been investigated in tobacco pollentubes as well as in leaf epidermal cells (Cheung et al., 2002). AGFP fusion of NtRab2 in pollen tubes was shown to locate

Fig. 7. Effect of wild-type and mutant forms of plant Rab1 and Rab2 proteins on ER-to-Golgi transport as illustrated by the use of various GFP markers.(a–c) Low-magnification CLS micrographs of tobacco leaf epidermal cells transformed with a secreted GFP marker, secGFP, alone (a) and alongside AtRab1b(b) or AtRab1b(N121I) (c). Epidermal cells of tobacco leaf areas infiltrated with secGFP alone (a) or secGFP and AtRab1b (b) show little or no intracellularfluorescence. Intracellular secGFP fluorescence is clearly visible when leaf areas are co-transformed with AtRab1b(N121I) (c). (d,e) High-magnificationCLS micrographs showing that, when co-expressed with AtRab1b(N121I), the pattern of intracellular secGFP fluorescence (d) resembles the fluorescencepattern caused by expression of GFP-HDEL, a soluble ER marker (e). (f,g) CLS micrographs of tobacco leaf epidermal cells expressing the Golgi marker ST-GFP and after 8 h recovery in water from BFA treatment in the absence (f ) or in the presence of AtRab1b(N121I) (g). In comparison with the control cell (f ),BFA recovery is inhibited by the presence of AtRab1b(N121I), as indicated by the ER-like pattern of ST-GFP fluorescence (g). (h–j) Fluorescence micro-graphs of tobacco pollen tubes expressing the Golgi-marker ERD2-GFP alongside wild-type and mutant forms of NtRab2. When co-expressed with wild-type NtRab2, ERD2-GFP locates to Golgi stacks (h), whereas co-expression with NtRab2(S20N) (i) or NtRab2(N119I) ( j) reduces the Golgi location ofERD2-GFP. Micrograph (i) courtesy of A. Cheung. (k,l) Fluorescence micrographs of tobacco pollen tubes expressing the plasma membrane marker Aha1-GFP alongside wild-type NtRab2 (k) or NtRab2(S20N) (l). When co-expressed with NtRab2, Aha-GFP labels the plasma membrane (k), whereas co-expressionwith NtRab2(S20N) leads to intracellular accumulation of Aha1-GFP. Scale bars: a–c,f,g = 25 µm; d–e,k = 10 µm; h,j,l = 20 µm. (a) to (e) are from Batokoet al. (2000) (fig. 4A,B). (h), ( j) and (l) from Cheung et al. (2002) (figs 4C,4E, 5D,E). Copyright the American Society of Plant Biologists and reprinted withpermission. (f ) to (g) are from Saint-Jore et al. (2002) (fig. 8g,h). Copyright Blackwell Publishing, Oxford, U.K., and reprinted with permission.



to BFA-sensitive punctate structures, identified as individualGolgi stacks by immunogold TEM with polyclonal anti-GFPserum. GFP fusions of two dominant-negative inhibitorymutants, GFP-NtRab2(S20N) and GFP-NtRab2(N119I),located to the pollen tube cytosol. Untagged versions of thetwo mutant proteins inhibited the transport of Golgi-resident,plasma membrane, and secreted GFP marker proteins to theirusual destination in pollen tubes (Fig. 7h–l). For instance,

the ERD2-GFP was shown to change from a predominantlyGolgi location to the ER when co-expressed with thedominant-negative inhibitory mutants of NtRab2 (Fig. 7h–j).NtRab2(S20N) and NtRab2(N119I) also had an inhibitoryeffect on pollen tube growth. Thus, Cheung et al. (2002)proposed a role for NtRab2 in ER-to-Golgi traffic. Interestingly,in contrast to pollen tubes, the GFP fusion of the wild-typeNtRab2 did not target Golgi stacks in tobacco leaf protoplasts

Fig. 8. Location of wild-type and mutant forms of Rab proteins involved in post-Golgi transport. (a) CLS micrograph of tobacco BY-2 cells simultaneouslyexpressing GFP-Pra2 (green image) and RFP-Pra3 (red image) (Inaba et al., 2002). The merged image shows that the two closely related Ypt3/Rab11homologues locate to different organelles of the endomembrane system. Micrograph courtesy of Y. Nagano. (b) CLS micrograph of Arabidopsis protoplastsexpressing Ara6-GFP, a homologue to mammalian Rab5, and labelled with the putative endocytic marker FM4-64. Mobile punctate structures andspherical organelles labelled with GFP (green image, arrowheads) were also labelled with FM4-64 (red image, arrowheads), as shown by the mergedimage (arrowheads). (c) CLS micrograph of Arabidopsis protoplasts expressing Ara6Q93L-GFP and labelled with the putative endocytic marker FM4-64.Ara6Q93L-GFP predominantly locates to the tonoplast and to spherical structures larger than those labelled by wild-type Ara6-GFP (green image; comparewith b). These spherical structures were also labelled by FM4-64 (red and merged images). N, nucleus. Scale bars: b–c = 10 µm. (b) and (c) are reprinted,with permission, from Ueda et al. (2001) (figs 6D,E,F and 7M).



and epidermal cells. However, a GFP fusion of an ArabidopsisRab2 homologue, AtRab2a (now called AtRabB1b), successfullytargeted the fusion to Golgi stacks in tobacco leaf epidermalcells (Neumann et al., 2003). This may suggest a role ofAtRab2a in a traffic event between the Golgi and a downstreamcompartment (U. Neumann, I. Moore, C. Hawes and H. Batoko,unpublished observations).

5.2. Regulatory proteins of post-Golgi transport steps

5.2.1. Rab11 (and Rab25) homologues. GFP-based studies onplant homologues of the mammalian Rab11 subclass suggestthat closely related Rab proteins locate to different compart-ments and might fulfil different functions (Inaba et al., 2002;Fig. 8a). GFP fusions of Pra2 and Pra3, two Pisum sativumRab11/Ypt3 homologues, have been located to punctatestructures in stably transformed tobacco BY-2 cells. WhereasGFP-Pra3 fluorescence was not affected by BFA treatment, thepunctate fluorescent GFP-Pra2 structures relocated to ER-reminiscent membranes after BFA treatment, suggesting thatGFP-Pra2 is targeted to Golgi stacks. However, GFP-Pra2 alsolabelled BFA-insensitive structures thought to be endosomal-like compartments. Co-localization experiments of GFP-Pra3with a fusion between AtVTI11 – a marker of the ‘trans-Golginetwork’ (TGN) and the PVC – and RFP resulted in partialoverlap of the two fluorochromes, suggesting that GFP-Pra3locates to the TGN and/or the PVC.

5.2.2. Rab5 homologues. GFP-based technology has also high-lighted functional diversification among plant homologuesof mammalian Rab5 GTPases (Ueda et al., 2001; Sohn et al.,2003; Bolte et al., 2004). The location of GFP-tagged versionsof two Rab5 homologues, Ara6 and Ara7, was investigated inArabidopsis protoplasts (Ueda et al., 2001). Ara6 is a memberof a novel, plant-unique type of Rab GTPases that lacks theC-terminal region, essential for attachment to membranesand subcellular localization. Instead, these unique Rabsare modified at the N-terminus for N-myristoylation andpalmitoylation (Ueda et al., 2001). Ara6-GFP was shown to labelboth punctate and spherical structures (in addition to somePM and ER labelling in some cells), whereas GFP-Ara7, theconventional Rab5 homologue, was predominantly detectedon punctate structures and less often on spherical organelles.Dual-colour imaging showed that Ara6-GFP- or GFP-Ara7-positive structures were also labelled with the styryl dyeFM4-64, a putative marker of the endocytic pathway (Fig. 8b).GFP fusions of the GTPase-deficient mutants, Ara6Q93L-GFPand GFP-Ara7Q69L, both located to aggregates of large sphericalstructures (FM4-64 positive; Fig. 8c), to the tonoplast and smallpunctate structures. Whereas Ara6Q93L-GFP was occasionallydetected on the PM, GFP-Ara7Q69L never was. Ueda et al. (2001)conclude that both Ara6 and Ara7 locate to endosomal-likeorganelles and regulate membrane fusion in the early endocyticpathway. However, based on the slightly different location of their

GTPase-deficient mutants as well as on differences in expressionpattern and in the amino acid sequence of the effector domain,Ara6 and Ara7 might regulate fusion of different endosomalpopulations in Arabidopsis protoplasts (Ueda et al., 2001).

Another member of the plant-specific Rab GTPases lackingC-terminal isoprenylation has also been isolated from a salinity-tolerant plant, Mesembryanthemum crystallinum (Bolte et al.,2000). Termed m-Rabmc, it is closely related to the Arabidopsis homo-logue Ara6 and correspondingly undergoes N-myristoylation(Bolte et al., 2004). In contrast to results shown by Ueda andco-workers on Ara6, m-Rabmc appears to be involved in thevacuolar trafficking pathway (Bolte et al., 2004). m-Rabmc hasbeen co-located with markers for the prevacuolar compart-ment of the lytic vacuole (BP80, Pep12). These localizationstudies were carried out by immunolabelling or by a combinationof expression of m-Rabmc-CFP constructs and immunolabelling.Both approaches resulted in a co-localization of m-Rabmc onmore than 80% of prevacuole. It has also been shown that m-Rabmc co-located partially on the Golgi apparatus. Importantly,it has been demonstrated that m-Rabmc was implicated in thetransport of the soluble marker protein aleurain-GFP, which istargeted to the acidic vacuole (Bolte et al., 2004).

A similar role in trafficking of soluble cargo from the PVC tothe central vacuole was also suggested for the third Arabidopsisisoform closely related to mammalian Rab5, Rha1 (Sohnet al., 2003). In Arabidopsis protoplasts, the dominant-negativeinhibitory mutant Rha1[S24N] (either tagged with the smallepitope haemagglutinin, HA, or fused to RFP) was shown toinhibit transport of GFP fused to the soluble vacuolar markerssporamin (spo-GFP) and Arabidopsis aleurain-like protein(AALP-GFP) to the central vacuole, presumably at the level ofPVCs. Interestingly, RFP-Ara7[S24N] but not Ara6[S47N]-HA caused the same altered distribution of spo-GFP. Biochemicalmethods confirmed that vacuolar trafficking of spo-GFP andAALP-GFP is inhibited, but also revealed that spo-GFP andAALP-GFP are secreted into the culture medium under theinfluence of HA-Rha1[S24N] and RFP-Rha1[S24N], respec-tively (Sohn et al., 2003).

Thus, questions regarding the location and function of theplant Rab5 homologues have yet to be conclusively answered.

Concluding remarks

Within a short time, fluorescent proteins have become invalu-able tools for in vivo investigations of the plant secretory path-way dynamics and regulation. However, as with any powerfultechnique, GFP technology has to be used wisely to avoidartefacts and data misinterpretations. For example, the natureof expression systems, whether by virus, agrobacterium orother transformation methods, may involve protein over-expression. As such, it is important to bear in mind the possibleeffect that protein over-expression may have on the plantendomembrane system. Therefore, the value of controlson transformation levels and ensuring that the labelling with



GFP fusions is due to correct targeting should not be under-estimated. Over-expression of protein may lead to ‘over-spill’ toother cellular compartments including the Golgi, vacuole andtonoplast, or even distension of the ER tubules as seen in cellsover-expressing non-GFP-labelled protein (Crofts et al., 1999).In addition, GFP tagging of proteins may result in loss of aspecific function or, worse, acquisition of new function.

Another possible limit of GFP technology could arise fromthe production of such artefacts in reporter systems involvingmalfolding of proteins and malfunction. Furthermore, asfor any other fluorescent probe, the sensitivity of GFP and itsspectral variants to pH and proteolysis should be consideredwhen investigating different organelles. One example is thevacuole, where GFP may accumulate but its fluorescence maybe easily quenched by the acidic pH and specific experimentalprocedures may need to be adopted in order to visualize GFP

fluorescence (Tamura et al., 2003). Finally, yet importantly,the expression and fluorescence of a GFP reporter may varyamong different reporter systems (Di Sansebastiano et al.,2004).

It should also be noted that organelles highlighted by GFPthat may have similar appearance to others described in theliterature (e.g. a dot or a ring) might not necessarily be thesame. Therefore, attempts to establish the subcellular loca-tion of novel protein markers fused to GFP should always beaccompanied by co-localization studies with other fluores-cent markers that label specific compartments. Alternatively,immunogold with GFP antiserum applied to TEM should beused to investigate the nature of the GFP labelling at highresolution (see Fig. 9).

As for any other light-microscopy-based technique, GFPtechnology also has its limits with regard to suborganelle

Fig. 9. Comparison of the location of two different Golgi markers in tobacco BY-2 cells with the CLSM (a,c) and the TEM (d). Although both GFP fusionproteins, ST-GFP (Batoko et al., 2000; Saint-Jore et al., 2002) and GFP-XylT36 (Follet-Gueye et al., 2003) locate to punctate structures when seen with theCLSM (a,b, respectively), their location within the Golgi stack, as seen by immunogold labelling using polyclonal anti-GFP antibodies, is different. WhereasST-GFP locates towards the trans-half of the Golgi stack (b), GFP-XylT36 is restricted to medial cisternae (d). Scale bars: a = 50 µm; b = 20 µm;c,d = 100 nm. (c) and (d) are reproduced, with permission, from Follet-Gueye et al. (2003) (fig. 6A,B).



resolution. Figure 9 illustrates that two GFP fusion proteinslocating to the Golgi bodies in tobacco BY-2 cells show a differentlocation within the stack with the aid of electron microscopy.

Modifications of GFP resulting in variations in both emissionand excitation wavelengths (Haseloff et al., 1999), coupledwith the development of other fluorescent proteins such asthe recently described red fluorescent protein (DsRed, Matzet al., 1999), will permit two- or even three-colour detectionof organelles in vivo (reviewed in Brandizzi et al., 2002b). Theadvent of new photoactivatable proteins, such as the photo-activatable GFP (Patterson & Lippincott-Schwartz, 2002)and the kindling RFP (Chudakov et al., 2003), coupled withimprovements in imaging and analysis techniques, will offeran impressive armoury for the investigation and understandingof the dynamic nature of living plant cells.

Acknowledgements

We thank all those who have kindly provided micrographs:H. Batoko, A. Cheung, G. P. Di Sansebastiano, M. L. Follet-Gueye,I. Hwang, Y. Nagano, A. Nakano, A. Nebenführ, C. Saint-Jore,M. Takeuchi and T. Ueda. We also thank F. Siefritz andN. Leborgne-Castel for the generous gift of the NtAQP1 cDNAand of the BobTIP26-1::GFP construct, respectively. Weacknowledge the Biotechnology and Biological SciencesResearch Council for supporting the work undertaken inour laboratory. Finally, we would like to thank Chris Hawes forhaving been a very good friend and an excellent tutor duringour residency at Oxford Brookes.

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