Chromosoma (2004) 112:255–266DOI 10.1007/s00412-003-0271-3

R E S E A R C H A R T I C L E

Sarah Docquier · Vinciane Tillemans · Roger Deltour ·Patrick Motte

Nuclear bodies and compartmentalization of pre-mRNA splicing factorsin higher plants

Received: 30 October 2003 / Revised: 10 December 2003 / Accepted: 10 December 2003 / Published online: 23 January 2004� Springer-Verlag 2004

Abstract We studied the fine structural organization ofnuclear bodies in the root meristem during germination ofmaize and Arabidopsis thaliana using electron microsco-py (EM). Cajal bodies (CBs) were observed in quiescentembryos and germinating cells in both species. Thenumber and distribution of CBs were investigated. Tocharacterize the nuclear splicing domains, immunofluo-rescence labelling with antibodies against splicing factors(U2B00 and m3G-snRNAs) and in situ hybridisation (withU1/U6 antisense probes) were performed combined withconfocal microscopy. Antibodies specific to the Ara-bidopsis SR splicing factor atRSp31 were produced.AtRSp31 was detected in quiescent nuclei and ingerminating cells. This study revealed an unexpectedspeckled nuclear organization of atRSp31 in root epider-mal cells where micro-clusters of interchromatin granuleswere also observed by EM. Therefore, we examined thedistribution of green fluorescent protein (GFP)-taggedatRSp31 in living cells after Agrobacterium -mediatedtransient expression. When expressed transiently, atR-Sp31-GFP exhibited a speckled distribution in leaf cells.Treatments with a-amanitin, okadaic acid, staurosporineor heat shock induced the speckles to reorganize.Furthermore, we generated stable Arabidopsis transgenicsexpressing atRSp31-GFP. The distribution of the fusionprotein was identical to that of endogenous atRSp31.Three-dimensional time-lapse confocal microscopyshowed that speckles were highly dynamic domains overtime.

Introduction

Splicing of nuclear pre-mRNA takes place within amulticomponent ribonucleoprotein (RNP) complex calledthe spliceosome, which contains the small nuclearribonucleoprotein particles (snRNPs) U1, U2 and U4/U5/U6 as well as non-snRNP proteins (Kramer1996). Thenon-snRNP serine/arginine-rich splicing factors (SR pro-teins) are involved in multiple steps of constitutive andalternative splicing (Lorkovic and Barta2002). The SRproteins constitute a family of splicing factors that arehighly conserved throughout the metazoa (Grave-ley2000). In Arabidopsis thaliana a growing number ofSR proteins have recently been uncovered (Lopato etal.1996, 1999a, 1999b,2002; Lorkovic and Barta2002;Savaldi-Goldstein et al.2003).

Splicing factors frequently exhibit local accumulationin subnuclear domains (Boudonck et al.1999; Eils etal.2000; Dundr and Misteli2001; Platani et al.2002). Inmammalian cells, snRNPs have been detected as a diffusenucleoplasmic pool and within the so-called speckles (orsplicing-factor compartments) (Misteli2000; Sleeman etal.2001). In many cell types, snRNPs also accumulate insmall spherical nuclear structures termed coiled bodies,recently renamed Cajal bodies (CBs) (Boudonck etal.1999; Gall2000,2001; Acevedo et al.2002; Hebert etal.2002; Ogg and Lamond2002; Platani et al.2002). Cajalbodies are involved in coordinating the assembly ofsnRNPs (Lamond and Earnshaw1998; Lewis and Toller-vey2000; Darzacq et al.2002; Jady et al.2003). Byimmunofluorescence microscopy, SR proteins can beobserved to colocalize with snRNPs within the specklescorresponding in electron microscopy (EM) to interchro-matin granule clusters (IGCs) and perichromatin fibrils.Speckles appear as storage and assembly sites ofspliceosomal components and the relocation of SR factorsfrom speckles to actively transcribed genes is dependenton their phosphorylation status (reviewed in Misteli2000).

Despite considerable progress in the understanding ofthe compartmentalization of mammalian nuclei, thefunctional nuclear organization of plant cells remains to

Communicated by P. Shaw

S. Docquier and P. Motte contributed equally to this work

S. Docquier · V. Tillemans · R. Deltour · P. Motte ())Laboratory of Plant Cell Biology, Department of Life Sciences,University of Li�ge,Bld du Rectorat, 27 Sart Tilman, 4000 Li�ge, Belgiume-mail: patrick.motte@ulg.ac.be

be defined. Microscopic investigations have shown thepresence of different nuclear bodies and a compartmen-talization of spliceosomal components as well (Beven etal.1995; Shaw1996; Boudonck et al.1998,1999; Straat-man and Schel2001; Acevedo et al.2002; Cui and MorenoDiaz De La Espina2003). In the present study, we lookedat the nuclear organization of splicing factors duringgermination of Zea mays and A. thaliana, two evolution-arily distant species. The embryo emerging from quies-cence undergoes postembryonic vegetative developmentand the root meristem resumes growth first (Bew-ley1997). The quiescent nucleus resumes sequentiallyall its metabolic activities with the onset of seedimbibition (Deltour1985; Bewley1997). Germination isthus particularly convenient for observing putative mod-ifications in spliceosomal factor relocation. One of themost interesting observations is a surprising speckledorganization of the Arabidopsis SR splicing factoratRSp31 in differentiated root cells like trichoblasts andatrichoblasts. This was an unexpected result since IGCreservoirs of SR factors in mammalian cells have neverbeen described in plants. To study further the distributionof plant SR proteins, we analysed the dynamics ofatRSp31 fused to the green fluorescent protein (atRSp31-GFP) in living cells after Agrobacterium -mediatedtransient expression and in stably expressing Arabidopsistransgenics. This is the first detailed analysis of thedistribution and dynamics of a plant SR splicing factor;the nature of the speckles in plant cells is discussed.

Materials and methods

Plant germination and growth conditions

Maize ( Z. mays L. var. Orellana and var. Dione) and A. thaliana(ecotype Columbia) seeds were germinated in Petri dishes andincubated in a controlled temperature chamber in darkness at 16�C(maize) and in continuous illumination at 20�C ( Arabidopsis). Forimmunocytochemistry, Arabidopsis quiescent embryos were ob-tained by cutting the seed coats with a scalpel and peeling them off.For transient transformation, A. thaliana and Nicotiana tabacum(cv. Petit Havana) seeds were sown on soil in culture chamber andallowed to germinate and grow under long-day conditions (16 hlight/8 h darkphotoperiod) at ~20�C. Healthy Arabidopsis plantswith well-developed leaf rosette and 20 cm height tobacco plantswere used for Agrobacterium -mediated transient expression.

RNA extraction and reverse transcriptase-polymerasechain reaction (RT-PCR)

For transcript analysis, tissues were harvested and immediatelyfrozen in liquid nitrogen. Total RNA was extracted using theguanidium method. cDNA for RT-mediated PCR was generatedusing SuperScript II Reverse Transcriptase (Invitrogen) accordingto the manufacturer’s instructions. The primers used for RT-PCRanalysis were as follow: for maize actin 1 (Accession no. J01238)(50-ATGGCTGACGAGGATATCCAGCC-30) and (50-GAAGCA-CTTCATGTGGACAATGCC-30); for HAT-B-p50 (Accessionno. AF171927) (50-ATGGCGCTCAAGCAGAAGGAC-30) and(50-TTGAACCACCAGCTCTGTCATC-30); for B1-type cyclin(CycZme1, Accession no. U66607) (50-ATGGAGTGCGCAAG-GGAGTTG-30) and (50-ACCTGACAACAGCTTCTTGTCAG-30).The PCR products were visualized on 1.0% agarose gels using a

UV light transilluminator. To confirm the RT-PCR specificity, thegenerated PCR products were subcloned into pGEM-T (Promega)and subsequently sequenced.

Expression vectors and polyclonal antibody production

The coding sequence of atRSp31 (Accession no. X99435) (Lopatoet al.1996) was amplified by RT-PCR using a 50 primer (50-ACATGCATGCGGCCAGTGTTCGTC-30) and 30 primer (50-GAAGATCTAGGTCTTCCTCTTGGGAC-30) containing Sph Iand Bgl II restriction sites (italicised), respectively. To producepolyclonal antibodies against atRSp31, the PCR product wassubcloned into the bacterial expression vector pQE70 (Qiagen) andnamed pQE-atRSp31 for recombinant protein production. Theconstruct was verified by DNA sequencing. The fusion proteincontaining a C-terminal (His)6 -tag was overexpressed in theEscherichia coli XL1Blue strain and purified according to themanufacturer’s manual using Ni-NTA metal-affinity resin (Qia-gen). Three rabbits were immunized with the purified (His)6 -tagatRSp31. In order to test the specificity of the sera, immunoblottingwas performed. Plant proteins were extracted in 0.1 M TRIS-HCl,pH 8.3, 0.5 M NaCl, 5 mM dithiothreitol and 5 mM EDTA,containing protease inhibitor (2 mM phenylmethylsulphonyl fluo-ride). Protein extracts were separated by SDS-polyacrylamide gelelectrophoresis and blotted on PVDF membrane (Roche). Immu-noblots were successively incubated with protein A purified IgG oraffinity purified antibodies (Burke et al.1982): anti-atRSp31 at a1:500 dilution and anti-rabbit IgG conjugated with alkalinephosphatase (Sigma) at a 1:3000 dilution followed by the detectionof proteins using 5-bromo-4-chloro-3-indolyl phosphate-nitrobluetetrazolium reagents (Roche).

Binary vector construction

For construction of the pBAR35S::atRSp31-GFP plasmid, theatRSp31 and EGFP full-length coding sequences were first PCRamplified using Pfu polymerase (Promega) from pQE-atRSp31 (seeabove) with the 50 primer (50-ATGAGGCCAGTGTTCGTCGGC-30) and the 30 primer (50-GAAGATCTTCCGGAAGGTCTTC-CTCTTGG-30) containing a Bgl II site (italicised) and pEGFP(Clontech; Accession no. U76561) with the 50 primer (50-GA-AGATCTATGGTGAGCAAGGGCGAGGAG-30) containing a BglII site, and the 30 primer (50-TTACTTGTACAGCTCGTCCATGC-30), respectively. The resulting PCR fragments were digested withBgl II and ligated. In this construction process, a linker of fouramino acids (Ser-Gly-Arg-Ser) was inserted between the atRSp31and GFP coding sequences. The chimeric gene was then amplifiedby using Pfu and cloned into the blunt-ended Sma I binary vectorpBAR-35S (Accession no. AJ251014, gift from Zs. Schwarz-Sommer, Max Planck Institute, Cologne, Germany), carrying aCaMV35S promoter/terminator. Independent clones were se-quenced to detect any mutations due to PCR. This construct anda control containing the EGFP sequence were used for transfor-mation.

Agrobacterium tumefaciens-mediated transient expression,drug and heat shock treatments

Transient expression in tobacco and Arabidopsis leaves wasperformed essentially as described by Batoko et al. (2000) usingA. tumefaciens strain GV3101 bearing the appropriate binaryvector. Transformed plants were then incubated under normalgrowth conditions as described above and leaf fragments weremounted in water 48–72 h after Agrobacterium infiltration andanalysed using laser scanning microscopy. The level of atRSp31-GFP expression under the control of the CaMV35S promoter wasremarkably stable and constant among transformed cells. Frag-ments of transformed leaves were used for drug treatments. Briefly,leaf tissue was submerged in, or floated on, 5 �M a-amanitin

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(Sigma), 5 �M okadaic acid (Calbiochem), 5 �M staurosporine(Sigma) or water as a control for different times (1, 3, 5 and 7 h).For heat-shock experiments, plants were placed in a culturechamber for 1 h at 42�C and leaf fragments were processed forconfocal imaging. Plants were then put back into the originalgrowth chamber.

Plant transformation and selection

For transformation, Arabidopsis plants were routinely grown in aculture chamber under short-day conditions (8 h light/16 hdarkphotoperiod) for 8–9 weeks and were then transferred tolong-day conditions. The in planta vacuum infiltration method wasperformed to obtain germinal transformants (Bechtold and Pelleti-er1998). After transformation, plants were grown under the sameconditions as prior to transformation. At least 20 plants weretransformed and seeds were collected from individual plants. T1transformants were obtained by selecting seeds for resistance toglufosinate-ammonium (50 �g/ml) (Riedel-de-Ha�n) on sterileplates. After 10 days of growth, glufosinate-resistant plants wereremoved from plates to be processed for observation of fluores-cence or planted directly into soil at the two-leafstage to obtain T2and T3 progeny.

Whole-mount in situ hybridization and fluorescence immuno-staining

Plasmid construction, preparation of U1 and U6 probes and whole-mount in situ hybridization were performed as described previously(Beven et al.1995; Boudonck et al.1998). Controls for in situhybridization were performed using sense riboprobes or RNase-treated (10 mg/ml RNase A) samples. The spliceosomal factorsU2B00 and atRSp31 were immunodetected using the mousemonoclonal 4G3 (Euro-diagnostica) and rabbit protein A purifiedIgG or affinity purified antibodies (Burke et al.1982) anti-atRSp31at a 1:100 dilution, respectively. For snRNA labelling, formalde-hyde-fixed samples were embedded in paraffin by standardprocedures and 5 �m deparaffined sections were incubated withthe primary mouse monoclonal trimethylguanosine cap antibodies(m3G, Euro-diagnostica). Secondary anti-mouse or anti-rabbitantibodies conjugated to fluorescein isothiocyanate (FITC) orAlexaFluor 546 (Molecular Probes) were used. As a control forimmunofluorescence, samples were incubated in the presence ofeither the pre-immune serum or the secondary antibody only.Nuclei were stained with 50 ng/ml 40,6-diamidino-2-phenylindole.Samples were mounted with an antifade medium and observed withthe confocal microscope.

Confocal imaging

The spatial distribution of immunolabelling and GFP fluorescencewere examined with a Leica TCS SP2 inverted confocal lasermicroscope (Leica Microsystems, Germany) equipped with argonand two helium-neon lasers, and an acousto-optical tunable filter(AOTF) for excitation intensity. Digitized images were acquiredusing a 63�NA 1.5 Plan-Apo water-immersion objective at1024�1024 pixel resolution. The diameter of the pinhole wasalways set up equal to the Airy unit. For each nucleus, serial opticalsections were recorded with a Z-step of ~0.2 �m. Images wereacquired under identical conditions and we ensured that themaximal fluorescence signal was not saturating the PMTs. Forfour-dimensional imaging, a XYZ multi-image series was definedby Z sectioning and Z series were then acquired manually every10 min for 90 min. Series of optical sections were either projectedas average/maximal projections or as stereo-pair using Leicasoftware (version 2.5). Captured images were exported as TIFFformat files and further processed using Adobe Photoshop.

Electron and immunoelectron microscopy

For EM, seeds and root fragments were fixed in 2% glutaraldehydein phosphate buffer, pH 7.4 and subsequently processed for Eponembedding. Ultrathin sections were observed with a Zeiss EM900electron microscope. For the three-dimensional (3D) reconstruc-tions of Arabidopsis nuclei, serial ultrathin sections were collectedon single-slot grids. For quantitative analysis of nuclear bodies, atleast three meristems per germination time were evaluated and, permeristem, 160 nuclear sections and 100 nucleolar sections wererandomly observed for counting the dense bodies (DBs) and CBs,respectively. The minimal number of sections to be considered wasdetermined for each time point by counting the nuclear bodies in anincreasing number of sections until their mean number becamestable. The distributions of the numbers of nuclear bodies werecompared by the Kruskal-Wallis non-parametric method. Allstatistical data were processed using Statistica 6.0 (Statsoft). Pre-embedding immunodetection was performed as follow: seeds androot meristems were fixed in 4% formaldehyde, washed in PEM,0.2% NP40 (50 mM PIPES, KOH, pH 6.9; 5 mM EGTA; 5 mMMgSO4.) and subsequently digested for 2 h with driselase (1%),cellulase (0.5%), pectolyase (0.025%). After washing in PEM,0.2% NP40, samples were treated with DMSO (0.5%). Cryosec-tions approximately 90 �m thick were made and allowed for ablocking step in PEM, 0.2% NP40, 3% BSA. Sections weresuccessively incubated at 4�C overnight with mouse monoclonal4G3 antibodies at a dilution of 1:100 and anti-mouse antibodiesconjugated to 10 nm gold particles at a 1:100 dilution. Sampleswere then embedded in Epon for EM observation.

Results

Nuclear bodies are observed by EM

The ultrastructure of maize and Arabidopsis root nucleihas been observed at different time points duringgermination and early seedling development. Comparisonof nuclear architecture did not reveal gross structuraldifferences between maize and Arabidopsis. Severalaspects of nuclear ultrastructural changes during germi-nation of maize and Sinapis alba have previously beendescribed in detail, except for the nuclear bodies on whichwe focused our present observations (Deltour et al.1986).At each germination time, nuclear bodies were observedin both species (Fig. 1 and not shown). Cajal bodiesexhibited a fibrillar texture and were localized eitherinside a slight depression on the surface of the nucleolusor free in the nucleoplasm, referred to, respectively, asnucleolus-associated CBs (NCBs) and free CBs (FCBs)(Fig. 1). Up to 72 h of germination, CBs appeared mainlyassociated with the nucleolus while after this period theywere most often free within the nucleoplasm (Figs. 1, 2).Both their morphology and localization appeared identicalbetween species.

In maize, DBs were easily distinguishable from CBsby their structure and smaller diameter. They were veryrare in quiescent nuclei but their number progressivelyincreased during germination with the resumption ofnuclear activity (Fig. 1). Most often, they have beenobserved free in the nucleoplasm but were sometimesobserved in close association with the nucleolus (Fig. 1).They displayed a dense fibrillar structure forming acompact spherical body, sometimes showing a crown of

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dense material. Although often seen as single, thesebodies were occasionally seen in groups of two or three(Fig. 1F). No dense bodies were observed in Arabidopsisas confirmed by our 3D reconstruction of nuclei (notshown). Their absence in Arabidopsis at any germinationtime could suggest species-specific functions. Furtherstudies will be needed to identify specific moleculeslocated in DBs and to gain insight into their possible rolein nuclear processes. The ribonucleoprotein nature of CBsand DBs was strongly suggested by the persistence oftheir contrast after an EDTA-regressive staining tech-nique that revealed ribonucleoprotein material around

bleached chromatin clumps and individual or smallgroups of granules in the interchromatin region (Fig. 1G).

Nuclear body numbers were analysed in maize mer-istematic cortical cells of the primary root duringgermination using EM (Fig. 1H). No important fluctua-tion of total CBs per nucleus was observed during the first72 h. However, discrimination between FCBs and NCBsshowed a slight increase and decrease, respectively, intheir number. These variations were further accentuatedafter this germination time. Interestingly, the significantdecrease of CB frequency was the direct consequence ofthe decrease in NCB frequency while the FCB frequencysignificantly increased. The frequency of DBs increased

Fig. 1 A–D Electron micro-graphs of cortical root cells ofZea mays at different timepoints during germination. Inquiescent cells ( A) and at 8 hafter imbibition ( B) crescent-shaped nucleolus-associatedCajal bodies (NCBs, arrows)are located at the nucleolarperiphery. After 24 h of germi-nation ( C), CBs become morespherical and after 120 h ( D),they appear in the nucleoplasmas well (free CBs, FCBs). Highmagnification of one CB ( E)and of dense bodies (DBs, F). GEDTA-regressive staining of afully active maize nucleusshowing dispersed granules be-tween and highly contrastedfibrils around bleached chro-matin clumps. H Frequencydistributions of the numbers oftotal CBs, NCBs, FCBs andDBs. Different letters on thetops of columns indicate statis-tical differences at P

by 20-fold during the first 72 h of germination (from 1.9to 38.1%). The results of the statistical analysis are shownin Fig. 1.

Splicing factor localization during germination

As U2B00 protein is a good marker of CBs in plants(Beven et al.1995; Boudonck et al.1998,1999; Acevedo etal.2002; Cui and Moreno Diaz De La Espina2003), itsdistribution was analysed by immunocytochemistry onboth maize and Arabidopsis root meristematic cells byconfocal microscopy. The labelling appeared very similar,if not identical, and Fig. 2A shows only the results ofU2B00 staining in maize nuclei due to space limitation. Inquiescent cells, U2B00 localized as weak punctate nucle-oplasmic labelling and a confocal Z-series through nucleishowed that CBs observed by EM were not labelled. At8 h, interestingly, U2B00 showed a heterogeneous nucle-oplasmic distribution and became highly enriched in pre-

existing CBs in both species as confirmed by immuno-electron microscopy (Fig. 2B). This latter technique alsoshowed a weak density of particles in the nucleoplasmand no labelling over the nucleolus, cytoplasm or DBs(not shown). Later, U2B00 was dispersed more homoge-neously throughout the nucleoplasm and concentrated inat least one fluorescent spot per nucleus (Fig. 2A). Thefoci associated with the nucleolus were generally largerthan the free ones.

The distribution of snRNAs observed using anti-m3GsnRNA antibodies was similar to the U2B00 proteindistribution in both species (Fig. 2A and not shown).One slight difference was a labelling of the CBs inquiescent nuclei. SnRNAs being among the best-charac-terized plant splicing factors, genes encoding U1 and U6have been cloned and in situ hybridization was performedwith anti-sense RNA probes during germination. Again,U1 and U6 probes showed similar distribution patterns inboth maize and Arabidopsis. U1 and U6 could be detectedin quiescent nuclei as shown in Fig. 2C. In active cells,

Fig. 2 A Distribution of U2B00and of m3G snRNAs in nucleifrom maize root tip meristemsat different time points duringgermination. The antigens arelocalized as a weak punctatenucleoplasmic labelling in qui-escent nuclei ( Q) and display amore homogeneous pattern withtheir concentration in brightspots corresponding to CBswith the onset of germination.Bars represent 8 �m. B U2B00labelling of the CBs at 8 and120 h of germination, respec-tively, visualized by electronmicroscopy. Bars represent200 nm. C In situ localization ofU1 and U6 snRNAs in quies-cent ( Q) and active nuclei. Barsrepresent 8 �m. D Immunoblotanalysis of bacterially expressedproteins (1, 2) and of totalproteins prepared from Ara-bidopsis inflorescences (4, 5)with anti-atRSp31 antibodies(1, 4) or pre-immune serum (2,5). 3 Coomassie blue-stainedgel shows the overall proteincomposition from inflores-cences. E Distribution of atR-Sp31 in nuclei fromArabidopsis root tip meristemsduring germination and in tri-choblast ( tricho.). Bars repres-ent 4 �m

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the strongest nuclear signal was localized as bright smallfoci for U1 and a more homogeneous bright interchro-matin network for U6. No labelling of nuclear bodies wasobserved in quiescent and active nuclei. In differentcontrols for immunocytochemical protocols and in situhybridization, no labelling was seen over nuclei (notshown).

To investigate the subcellular distribution of a plant SRsplicing factor, rabbit polyclonal antibodies were raisedagainst atRSp31, which is characterized by the presenceof one N-terminal RRM and a C-terminal RS domain(Lopato et al.1996). The specificity of the affinity-purified antibodies was tested by immunoblot analysison a protein extract from E. coli and from Arabidopsisplants (Fig. 2D). It showed that immune rabbit serumraised against recombinant atRSp31 specifically recog-nized His-atRSp31 in the crude extract of E. coli(Fig. 2D). With the protein extract from Arabidopsisplants, this antibody stained a single band at 31 kDa,corresponding to the expected molecular mass of full-length atRSp31. The pre-immune serum from the samerabbits gave no signal (Fig. 2D). Thus, our polyclonalantibody is highly specific to the Arabidopsis atRSp31factor. Using these antibodies in immunofluorescenceconfocal microscopy, we analysed the subcellular distri-bution of atRSp31 in meristematic cortical root cellsduring the early germination of A. thaliana (Fig. 2E).AtRSp31 was detected as punctate foci in quiescentnuclei. With the onset of nuclear reactivation as early as8 h after seed imbibition, the foci organization of atRSp31became less apparent and a more homogeneous distribu-tion was observed in cortical cells of the root meristem(Fig. 2E). However, a speckled distribution of atRSp31was observed in the nucleoplasm of trichoblasts andatrichoblasts in the elongation and differentiation zones(Fig. 2E). Our anti-atRSp31 sera were tested on maizeembryos but no labelling was detected (not shown),showing the high specificity of the anti-atRSp31 antibod-ies.

RT-PCR analyses during germination

The overall modifications in the distribution of splicingfactors during early germination suggest that the splicingprocess could slowly and/or sequentially resume. In orderto verify such a possible sequential resumption, welooked for intron-containing genes in databases that couldbe expressed as early as seed imbibition and determinedtheir transcript profile during early germination. Weperformed RT-PCR analysis with terminal cDNA primersof actin, HAT and cyclin on total RNA extracted frommaize root tips. As shown in Fig. 3, PCR of actin yieldedtwo bands corresponding to the sizes of the unspliced pre-mRNA and spliced mRNA (2.201 and 1.128 kb, respec-tively); PCR of cyclin and HAT yielded single bands of1.437 kb and 1.404 kb, respectively, corresponding to thesizes of spliced mRNAs. RT-PCR was carried out ondifferent maize varieties and the only difference was the

PCR products for HAT and actin appearing as singlebands of correctly spliced mRNAs in quiescent embryos(not shown). The observation that unspliced pre-mRNAsand spliced mRNAs coexist in quiescent embryossuggests that transcription and splicing could be disso-ciable processes in some physiological situations.

Localization of atRSp31 after transient expression,drug treatment and heat shock

The experiments described so far examined the spatiallocalization of splicing factors by imunocytochemistry onfixed tissues, which did not assess the dynamics of anyfactor. To examine further the subcellular localization anddynamics of atRSp31 in living plant cells, we generated atranslational fusion construct of this splicing factor withGFP linked to the C-terminus of atRSp31 under thecontrol of the CaMV35S promoter. First, we usedtransient expression to develop a rapid and efficient assayfor the in vivo spatial organization of plant SR splicingfactors. Fluorescence of GFP-tagged atRSp31 was ob-served in Arabidopsis and tobacco leaf cells afteragroinfiltration-based transient expression. We ensuredthat agroinfiltration and subsequent fluorescent analysisdid not induce cell death or necrosis. Moreover, we didnot observe any toxic effects of atRSp31-GFP intransiently overexpressing cells. The Agrobacteriuminoculation only induced weak chlorosis in tobacco,which facilitated the identification of the transientlyexpressing cells. The fluorescence of GFP could be easily

Fig. 3 Transcript analysis of the expression of actin ( A), HAT ( B)and cyclin ( C) during early germination of Zea mays. Ethidiumbromide-stained gels showing products of the reverse-transcribedpolymerase chain reaction (RT-PCR). Lanes 2–7 Products frommaize RNA amplified at different time points of germination. Unitsare in kilobases. Lane M markers

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detected by epi-fluorescence using an FITC filter block.Many cells were observed to express the fluorescentproteins as early as 24 h after infiltration. The expressionlevel reached an optimum after 48 h and the fluorescencewas very stable and could still be detected for at least 5–6 days in both species although the efficiency of themethod was always higher in tobacco (not shown). Aspreviously visualized using anti-atRSp31 antibodies andindirect immunofluorescence, the speckled pattern ofatRSp31-GFP was clearly apparent in both tobacco andArabidopsis leaf cells (Fig. 4). To our knowledge, this isthe first report of the efficient transient transformation ofArabidopsis leaves after Agrobacterium inoculation. Thespatial distribution of free GFP was observed byexpressing a binary vector containing only the GFP genedownstream of the CaMV35S promoter. As expected

from published data, expression of free GFP producedhigh and homogeneous fluorescence in both cytoplasmand nucleus due to passive diffusion through nuclearpores (Fig. 4A). No speckled accumulation of GFP ineither cytoplasm or nucleus was observed (Fig. 4).Confocal Z-series of tobacco and Arabidopsis nucleiclearly demonstrated a speckled fluorescence organiza-tion of atRSp31-GFP in the entire nuclear depth (Fig. 4B).The analysis of GFP fluorescence at different time pointsafter agroinfiltration showed that nuclear speckles ap-peared as early as fluorescence could be detected (~24 h),suggesting that subnuclear distribution of atRSp31-GFP isrelatively independent of its overexpression and overac-cumulation.

Inhibition of RNA polymerase II transcription by a-amanitin caused the disappearance of the diffuse fraction

Fig. 4A–C Agrobacterium -mediated transient expression ofatRSp31-GFP (green fluores-cent protein) fusions or freeGFP under the control of theCaMV 35S promoter. A Leftpanels: low-magnification con-focal microscopy through epi-dermal cells expressingatRSp31-GFP and free GFPmerged with chlorophyll aut-ofluoresence ( bars represent80 �m). Middle and right pan-els: high resolution confocalmicroscopic sections show dis-crete nuclear speckles in tobac-co and Arabidopsis leaf cellsexpressing atRSp31-GFP ( barsrepresent 4 �m). Relative in-tensity fluorescence profiles areplotted along the line across thenucleus. B Selection of serialoptical sections through the nu-cleus of an atRSp31-GFP ex-pressing Arabidopsis leaf cell.In addition to diffuse fluores-cence, speckle-like structuresare apparent throughout the nu-cleus. Bar represents 2 �m. CAtRSp31-GFP localization afterdrug treatments (a-amanitin,okadaic acid and staurosporine)and heat shock. ( Nu nucleolus.)Bar represents 2 �m

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of atRSp31-GFP and its redistribution into larger nucleardomains (Fig. 4C). The use of okadaic acid andstaurosporine (Ser Thr phosphatase and kinase inhibitors,respectively) on transformed leaves provoked a similardisappearance of the diffuse atRSp31-GFP and its con-

centration into bigger speckles and around the nucleolus(Fig. 4C). Heat shock treatment, known to disruptspliceosomal components, led to accumulation of atR-Sp31-GFP around the nucleolus as well (Fig. 4C).

Fig. 5 A–E Confocal micro-scopic analysis of a transgenicseedling expressing atRSp31-GFP. Bright-field image ( A)and in vivo analysis of GFPfluorescence ( B) of a primaryroot expressing the fusion pro-tein. The maximal projectionshows the nuclear localizationof atRSp31-GFP in every cell ofthe root. High resolution imagesof nuclei from one meristematiccell ( C), one atrichoblast cell ofthe elongation zone ( D) andone trichoblast cell of the dif-ferentiation zone ( E). F–HFixed-trichoblast cell express-ing atRSp31-GFP ( green) im-munostained with antibodiesagainst U2B00 ( red). The over-lap ( H) shows distinct nuclearlocalization of the speckles andCajal body ( arrow). I–L Ul-trastructure of nuclei of wild-type meristematic (I) and tri-choblast (J) cells and of trans-genic meristematic (K) andtrichoblast (L) cells. In tri-choblast cells, some microclus-ters of interchromatin granulesare observed (see insert for highmagnification). Bars represent200 �m ( A, B); 1 �m ( C–L)

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Dynamic nuclear localization of atRSp31 in Arabidopsis

Since a speckled organization of the atRSp31 SR splicingfactor occurred after our transient expression experiments(strongly suggesting a functional localization of the fusionprotein), we generated transgenic Arabidopsis plantsstably expressing the atRSp31::GFP fusion construct,allowing us to compare different cell types and develop-mental stages. Similar results were obtained with manyindependent lines (not shown). A low-magnificationfluorescence analysis showed a clear nuclear localizationof the fusion protein in the whole seedling root (Fig. 5).As already reported (Chytilova et al.2000), nuclei withinroot-hair cells were observed to have a complex non-spherical shape. In meristematic cells, uniform fluores-cence was observed with no apparent accumulation ofatRSp31-GFP in foci (Fig. 5C). The most impressivespeckled distribution of GFP-tagged atRSp31 could beobserved in epidermal cells (both atrichoblasts andtrichoblasts), similar to what we observed using antibod-ies (Figs. 5, 6). Interestingly, the analysis of transgenicsand of transient transformation demonstrated that over-expression of atRSp31-GFP resulted in its accumulationin speckles in a particular cell or tissue type. We nextsought to observe whether overexpressed atRSp31-GFPaccumulated in CBs. U2B00 immunostaining in atR-Sp31GFP-expressing cells displayed a diffuse U2B00

distribution, with additional bright CBs. The nucleoplas-mic diffuse labelling of U2B00 and atRSp31-GFP colo-calized but U2B00 protein did not accumulate in thespeckles (Fig. 5F–H). Next, we legitimately wonderedwhether the overexpression of atRSp31-GFP under thecontrol of CaMV35S promoter could modify the organi-zation of nuclei. In other words, could overexpressedatRSp31-GFP induce the development/formation of IGCs

in plant cells? High-resolution nuclear organization ofArabidopsis transgenics was investigated by EM andcompared with that of wild-type seedlings. Observationswere made in both meristematic and differentiation zonesof the root. No gross changes in overall nuclearultrastructure were observed between T2/T3 transgenicsand wild-type, either in meristematic cells or in thedifferentiation zone (Fig. 5I–L). Although smaller than inmammalian cells, micro-clusters of IGs were neverthelessobserved in trichoblasts (Fig. 5J, L).

Since the dynamic properties of speckles in plant cellsare largely unknown, high-resolution 3D time-lapseimaging of atRSp31-GFP in T2 and T3 seedlings wasperformed (Fig. 6). Four-dimensional analysis of meri-stematic nuclei did not show any modification in thefluorescence pattern over a long period (P. Motte,unpublished data). However, in atrichoblasts, the stereoimages presented in Fig. 6 show complex movement andshape modification of atRSp31-GFP over a long period.Although the nuclear speckles of atRSp31-GFP remain inrelatively stable spatial domains during a time-lapseanalysis of more than 90 min, their periphery coulddisplay a likely non-random budding and fusing. Rapidmovement (a few seconds) of atRSp31-GFP was alsovisible at the periphery of the speckles (P. Motte,unpublished data). Unfortunately, four-dimensional anal-ysis of root-hair nuclei was difficult to perform because ofthe rapid and constant movements of nuclei along thecells during laser scanning (P. Motte, unpublished data).

Fig. 6 Four-dimensional imag-ing of an atrichoblast nucleusshowing movement, fusion andbudding of atRSp31-GFP overtime ( arrows). Z series wereacquired at ~10 min intervalsand three-dimensional stereo-pair images were reconstructedfor each time point. The time inminutes is indicated at the topof each section. Bar represents2 �m

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Discussion

Nuclear bodies

The nucleus is now recognized as a complex organellewith spatially organized territories generically referred toas nuclear bodies. With a diameter ranging from less than1 �m to more than 10 �m, CBs have been identified inboth animal and plant cells and appear as highly dynamicstructures (Beven et al.1995; Boudonck et al.1998,1999;Jennane et al.1999; Matera1999; Lewis and Toller-vey2000). In this work, CBs were observed in quiescentand germinating cells in both species. In quiescent nuclei,CBs are very often in close association with the nucleolusand they do not label with anti-U2B00 antibodies or U1/U6antisense probes. This suggests either that splicing factorsare not strictly required for the assembly of CBs or thatsplicing factors can leave CBs (as a result of seeddesiccation) without affecting their morphology. Soonafter seed imbibition, pre-existing CBs became highlyenriched in U2B00. Their content modification correlateswith the resumption of nuclear reactivation during earlygermination. At 72 h of germination, a significantdecrease of total CBs with a concomitant decrease ofNCBs and an increase of FCBs were observed. Similarvariations in the number and localization of CBs fromdormant/quiescent to transcriptionally active cells havebeen reported in other plants (Beven et al.1995; Boudon-ck et al.1998,1999; Jennane et al.1999; Acevedo etal.2002; Cui and Moreno Diaz De La Espina2003). Inmaize, the decrease of CBs can be related to theresumption of mitotic activity in the meristem. Indeed,previous studies have established that the majority of thecells are in G1 and that the first wave of mitosis occurs atabout 72 h after imbibition (Deltour1985). This is in goodagreement with the results of Boudonck et al. (1999) whohave analysed the localization of GFP-tagged U2B00

protein in BY2 tobacco cells and Arabidopsis transgenics.In living plant cells they have observed movements ofCBs from the nuclear periphery to the nucleolus, theirfrequent fusion and a decrease in CBs per nucleus directlyafter cell division (Boudonck et al.1999).

Interestingly, we detected splicing factors in inactivequiescent nuclei. These factors were enriched in particularribonucleoprotein domains, as also described in dormantonion cells and named micro-speckles (Cui and MorenoDiaz De La Espina2003). As suggested by Cui andMoreno Diaz de la Espina (2003), quiescent micro-speckles could be storage sites of splicing factors,becoming biologically active and redistributed with theonset of nuclear activation. Although few data areavailable on both the nature and the localization of storedmRNAs in the quiescent embryos, these quiescent/dormant micro-speckles could alternatively representresidual preformed mRNA linked to processing factorsthat have survived the desiccation programme occurringat the end of seed maturation. Although we do not knowwhether polyadenylated RNAs are located within thesemicro-speckles, it has been reported that inhibition of

mammalian RNA polymerase II induced a population ofstable long-lived poly(A) RNA associated with pre-mRNA splicing factors forming IGCs (Huang etal.1994). Similarly, seeds becoming metabolically quies-cent are characterized by transcription that is slowlyarrested and preformed mRNAs could remain linked toprocessing factors in quiescent nuclei. Our RT-PCR datasuggest that quiescent long-lived mRNAs are correctlyspliced mRNAs, regardless of where these are located.

Speckles, or not speckles, in the plant nucleus:the beginning of an answer?

The compartmentalization of essential SR splicing factorshas been extensively studied in animal cells (Lamond andEarnshaw1998; Eils et al.2000; Misteli 2000 Allemand etal.2001; Dundr and Misteli2001). SR proteins concentratein precise subnuclear speckles consisting of many irreg-ularly shaped domains in the interchromatin compart-ment. In mammalian cells, speckles correspond to IGCscomposed of particles measuring 20–25 nm in diameterand perichromatin fibrils. Whereas a plethora of Ara-bidopsis SR splicing factors has been identified (Lorkovicand Barta2002), their precise subcellular distribution islargely unresolved. Since the Arabidopsis atRSp31 pro-tein has been shown to complement the HeLa S100extract (Lopato et al.1996), there was significant interestin investigating its localization and dynamic redistributionwithin the context of plant nuclear organization. Consis-tent with its role as a splicing factor (Lopato etal.1996,2002), atRSp31 protein was exclusively localizedin the nucleus by indirect immunofluorescence. More-over, atRSp31 factors displayed a heterogeneous speckleddistribution in root epidermis cells (trichoblasts andatrichoblasts). The restricted nuclear localization ofatRSp31, its diffuse distribution and accumulation in anumber of discrete speckles were further observed bytransient and stable expression of GFP-tagged atRSp31 inboth tobacco and Arabidopsis cells.

Although atRSp31 can display a speckled organiza-tion, the presence of IGCs in plant cells is still a matter ofdebate (Testillano et al.1993; Acevedo et al.2002; Cui andMoreno Diaz De La Espina2003). A clear IGC organi-zation such as the one described in mammalian cells hasnever been described in plant cells by EM although insome cell types, some small clusters of particles of 20–30 nm can be observed (Testillano et al.1993; Acevedo etal.2002; Cui and Moreno Diaz De La Espina2003; and thepresent report). Some questions can legitimately be raisedas to whether the GFP-tagged atRSp31 is fully functionalas an essential splicing factor and whether the speckledconformation of the fusion protein has some biologicalsignificance. Due to the strong constitutive CaMV35Spromoter, the accumulation of atRSp31-GFP in nucleicould indeed amplify (or create) the speckled organiza-tion of SR splicing factors. However, a functionalarrangement of atRSp31-GFP rather than an in vivoaggregate artefact due to protein overaccumulation is

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suggested by several arguments. First, in Arabidopsistransgenics (T1, T2 or T3), the most characteristicspeckled organization of atRSp31-GFP was observed inthe epidermis, where comparable immunofluorescencelabelling of the wild-type atRSp31 was detected in fixedroot tissues. Second, we did not observe nuclear specklesin root tips (e.g. meristematic zones) where intensefluorescence was nevertheless present, indicating overac-cumulation of atRSp31-GFP. Third, our time-lapse andfour-dimensional analysis of fusion protein dynamicsshowed budding movement at, and exchange between,speckle peripheries. Moreover, the redistribution ofatRSp31-GFP after heat shock and inhibitor treatmentssuggests that the fusion protein is functional and that thephosphorylation/dephosphorylation level and the tran-scriptional activity of the cell regulate its nuclearlocalization. We have observed that overexpressed mutantatRSp31-GFP proteins with deleted RS domains do notconcentrate in discrete subnuclear foci after transienttransformation, indicating that the RS domain is essentialin directing speckled organization in living plant cells (V.Tillemans and P. Motte, unpublished).

Our report also shows that agroinfiltration-mediatedtransient expression can provide a fast and powerful toolfor the study of the distribution of splicing factors in vivo.Moreover, transient expression in tobacco demonstratesthat the presence of endogenous Arabidopsis atRSp31 isdispensable for the speckled organization of atRSp31-GFP.

If we clearly show that Arabidopsis SR proteins arelocalized in nuclear speckled domains, we do not knowhow (or whether) they fit into the existing model ofcompartmentalization of mammalian SR splicing factors.We have to consider the possibility that the functions ofspeckles revealed by atRSp31 have yet to be identified inplants. Interestingly, speckle-like structures have beendescribed in plant nuclei for many developmental regu-lators as well, like phytochrome or CRY2 photoreceptors(Mas et al. 2000; Kircher et al. 2002; Kevei andNagy2003). The speckled distribution of these regulatoryproteins is suggested to correspond to macromolecularcomplexes functional in the synthesis and processing ofspecific target gene transcripts. The speckled organizationof plant SR protein might reflect a high and transientdemand of splicing factors in response to activelytranscribed genes rather than a reservoir of pre-mRNAprocessing factors shuttling between storage/preassemblysites to transcription sites. We can reasonably ask whetherspeckles and micro-clusters of IGs observed in Arabidop-sis epidermis is possibly linked to a high or lowtranscriptional activity of these differentiated cells. Thespeckled distribution of SR proteins in plants couldcorrespond to even larger macromolecular complexescoordinating spatial organization of active euchromatin(from chromatin remodelling to pre-mRNA processing),these hypotheses not being mutually exclusive.

Acknowledgements We are grateful to Dr P. G�rard (Universit� deLi�ge; Unit� de Recherche de Statistique — Aspects Exp�rimen-

taux) for assistance with statistical analysis, Prof. Marc Boutry(Universit� Catholique de Louvain, Belgium) for valuable advicewith transient transformation and to Prof. Peter Shaw (John InnesCentre, Norwich, UK) for helpful comments. This research wassupported by grants from “National Fund for Scientific Research”(grants no. 2.4547.99, 2.4520.02 and 2.4542.00) and from “FondsSp�ciaux du Conseil de la Recherche” of the University of Li�ge.S.D. was supported by an FRIA grant (Fonds de la Recherche pourl’Industrie et l’Agriculture) and V.T. is supported by FRIA.

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