Ai

Ja

Eb

c

A

a

ARRA

KCDMMNPPR

1

tpalt2ioo

M

0d

Journal of Plant Physiology 168 (2011) 746–757

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .de / jp lph

change of developmental program induces the remodeling of thenterchromatin domain during microspore embryogenesis in Brassica napus L.

.M. Seguí-Simarroa, P. Corral-Martíneza,1, E. Corredorb,1,2, I. Raskac, P.S. Testillanob, M.C. Risuenob,∗

Instituto para la Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, Ciudad Politécnica de la Innovación (CPI),dificio 8E – Escalera I, Camino de vera, s/n, 46022 Valencia, SpainPlant Development and Nuclear Architecture, Centro de Investigaciones Biológicas (CIB)-C.S.I.C, c/Ramiro de Maeztu, 9, 28040 Madrid, SpainInstitute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague and Department of Cell Biology, Inst. of Physiology,cademy of Sciences of the Czech Republic, v.v.i., Albertov 4, 128 00 Prague, Czech Republic

r t i c l e i n f o

rticle history:eceived 29 July 2010eceived in revised form 20 October 2010ccepted 21 October 2010

eywords:ajal bodiesifferentiationaturationicrospore embryogenesisucleusollenroliferationapeseed

a b s t r a c t

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental path-way towards embryogenesis producing multicellular embryos from which, finally, haploid and doublehaploid plants develop. The architecture of the well-organized nuclear functional domains changes inresponse to DNA replication, RNA transcription, processing and transport dynamics. A number of sub-nuclear structures present in the interchromatin region (IR, the nuclear domain between chromosometerritories) have been shown as involved, either directly or indirectly, in transcriptional regulation. Thesestructures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies(CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochem-ical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels tocharacterize the changes in the functional architecture of the nuclear interchromatin domain duringtwo developmental programs followed by the microspore: differentiation to mature pollen grains (tran-scriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly

engaged in transcription). Our results revealed characteristic changes in size, shape and distribution ofthe different interchromatin structures as a consequence of the reprogramming of the microspore, allow-ing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activitiesduring proliferation and differentiation events, and suggesting that RNA-associated structures could be aregulatory mechanism in the process. In addition, we document the presence of two structurally different

nd C

types of CBs, and of IGC adescribed in plants.

. Introduction

The microspore or immature pollen grain normally followshe gametophytic program and differentiates to form the matureollen, a process that can also be reproduced in vitro. Upon thepplication of a stress treatment, it can be deviated towards a pro-iferation process leading to embryogenesis and plant regeneration,he so-called microspore embryogenesis (Seguí-Simarro and Nuez,

008), a process which represents an important tool in plant breed-

ng to obtain double-haploid plants. The entry into proliferationf differentiating cells is a key event in plant development and inrganogenic and morphogenetic processes. Since the cell nucleus

∗ Corresponding author.E-mail address: risueno@cib.csic.es (M.C. Risueno).

1 These authors have contributed equally.2 Present address: Department of Genetics; Universidad Complutense de Madrid,adrid, Spain.

176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.jplph.2010.10.014

B-associated regions, similar to those present in animal cells, and not yet

© 2010 Elsevier GmbH. All rights reserved.

governs the cell activity, knowledge of the dynamics of the nucleardomains during the activation of such processes will provide newinsights into the mechanisms that regulate them. Pollen develop-mental programs represent model systems to analyze the cellularchanges that accompany the change of developmental program andthe switch to proliferation from plant differentiating cells.

The nucleus is the cell factory where DNA replication and RNAtranscription and processing take place. It is a highly dynamiccompartment where different activities are confined to specificdomains, whose structures are markedly dependent on theirfunction (Spector, 1993a). Nuclear chromatin is divided in chro-mosome territories, which encompass (1) condensed chromatin(heterochromatin, transcriptionally inactive) and (2) decondensedchromatin (euchromatin), where transcriptionally active genes are

located (Cremer and Cremer, 2001). These different levels of con-densation, together with other remodeling and epigenetic events,are considered as a first, pretranscriptional level of regulation ofgene expression (Jarillo et al., 2009). Indeed, rearrangements in theposition and condensation of chromosomes are known to play an

f Plan

iesppTmfltfc

idtopomCdsa

fri(2m1spppbTiaiants2maiamSd1sacRV

otdpos

J.M. Seguí-Simarro et al. / Journal o

mportant role in the establishment of the different developmentalxpression patterns by exposing and allowing access of the tran-criptional machinery to specific gene sequences. From a molecularoint of view, the different regulatory steps of transcription andre-mRNA processing in eukaryotes are well known processes.here is a wealth of data on how they proceed and the differentolecular players involved. Indeed, in vitro transcription is possible

or long, which has boosted the emergence of a variety of molecu-ar biology techniques. However, there is less information on whereranscription-related processes take place in vivo, and how the dif-erent structures involved change their architecture in response toell requirements during different developmental programs.

The nuclear space separating chromosome territories is thenterchromatin region (IR). At the boundaries of the IR with the con-ensed chromatin, decondensed chromatin fibers are transcribedo RNA in the form of perichromatin fibrils (PFs). PFs are short fibersf 3–5 nm in diameter, present in a dispersed pattern through theerichromatin region. They are thought to be the visible evidencef nascent pre-mRNA coupled to the co-transcriptional splicingachinery (Monneron and Bernhard, 1969; Testillano et al., 1993;

marko et al., 1999; Fakan, 2004). In addition, the IR houses theifferent structures involved in post-translational regulatory stepsuch as RNA processing and nuclear protein modification (Cremernd Cremer, 2001).

Once the pre-mRNA (hnRNA) sequence is generated in theorm of a PF, three subdomains are proposed as directly or indi-ectly involved in pre-mRNA splicing, processing and transport:nterchromatin granules, Cajal bodies and perichromatin granulesSpector, 1993a; Raska, 1995; Lamond and Spector, 2003; Fakan,004). Interchromatin granule clusters (IGCs) have been found inammalian (Puvion et al., 1984) and plant nuclei (Testillano et al.,

993), and they are seen as ‘speckles’ under the fluorescence micro-cope. They are interchromatin structures composed of 20–25 nmarticles embedded in a thin fibrillar matrix. Although differentre-mRNA splicing factors, including snRNPs and SR proteins, areresent in IGCs, no DNA or transcriptional activity has consistentlyeen detected in these structures (Thiry, 1995; Cmarko et al., 1999).hus, ICGs are currently considered as storage, assembly and mod-fication compartments for the delivery of transcription factors toctive transcription sites (Lamond and Spector, 2003). Cajal bod-es (CBs) are universal, multifunctional, nearly round, convolutednd highly dynamic structures frequently found associated to theucleolus. snRNPs, the p80 coilin protein, the U2AF splicing fac-or and diverse snRNAs have been detected in CBs, along witheveral nucleolar proteins such as fibrillarin (reviewed in (Gall,000; Lafarga et al., 2009). However, poly-A RNA, rRNA, nascentRNA transcripts, the SC-35 protein, DNA or RNA polymerase II

re not found in CBs, indicating that they are not directly involvedn transcription. Instead, it is believed that CBs are involved in thessembly and modification of the machinery for processing of pre-RNA, pre-rRNA and histone pre-mRNA (Gall, 2000; Matera, 2003;

haw and Brown, 2004). Different studies have related the abun-ance of CBs to cell activity (Andrade et al., 1993), type (Raska et al.,991) and developmental stage (Boudonck et al., 1998). Althoughmaller and less conspicuous, the perichromatin granules (PGs)re also integral components of the eukaryotic nucleus. RNA pro-essing, altered transport, storage and/or degradation of specificNAs is thought to be mediated by PGs (Puvion and Lange, 1980;azquez-Nin et al., 1997).

As evidenced in most of the literature cited above, knowledgef in vivo functional organization of the different subnuclear struc-

ures involved in transcriptional regulation has traditionally beenerived, to a great extent, from studies on animal nuclei. Thelant nucleus has been studied less. To contribute to knowledgef the relationship between plant nuclear remodeling and expres-ion patterns, in this work we analyzed the changes in nuclear

t Physiology 168 (2011) 746–757 747

architecture during two different developmental programs involv-ing proliferation and differentiation. We studied Brassica napusembryogenesis-induced microspores and maturing pollen grains.In microspores, we induced an embryogenic developmental pro-gram defined by an initial, proliferative phase with increasedtranscriptional activity (reviewed in (Seguí-Simarro and Nuez,2008). In parallel, we studied pollen differentiation, characterizedby the formation of a moderately active vegetative cell, and a gen-erative cell transcriptionally silent (Bednarska, 1984).

The comparative study of microspores and pollen grains com-mitted to proliferative and differentiation events constitutes agood model to study cellular changes related to a developmen-tal reprogramming (Seguí-Simarro et al., 2003, 2005; Barany et al.,2010). In this study, we focused on the changes pertaining to thenuclear structures of the interchromatin domain (perichromatinfibrils, interchromatin granules, Cajal bodies and perichromatingranules). Our results indicated that a remodeling of the inter-chromatin structures accompanied the change of developmentalprogram of the microspores, with those changes being related toproliferation and differentiation events. The results also reveal twonovel structural domains, not previously described in plants, andpoint to the notion that the different availability and abundance ofthese structures may serve as a regulatory mechanism operating ata post-transcriptional level.

2. Material and methods

2.1. Plant material

Brassica napus L. cv Topas donor plants were grown as previ-ously described (Seguí-Simarro et al., 2003). Microspore cultures,embryogenesis induction, and sampling at specific culture stagesfor light and electron microscopy, immunofluorescence, andimmuno-gold labeling were performed essentially according toSeguí-Simarro et al. (2003), with some modifications to promotethe suspensor-bearing embryogenic route as described (Supenaet al., 2008). For the study of pollen development, anthers at differ-ent developmental stages were selected and excised from plants.

2.2. Electron microscopy and ultrastructural cytochemistry

Samples to be observed for electron microscopy were fixedin Karnovsky fixative (4% formaldehyde + 5% glutaraldehyde in0.025 M cacodilate buffer, pH 6.7), post-fixed in 2% OsO4, dehy-drated in a methanol series for 3 days and slowly embedded inEpon resin for 2 days. Epon blocks were polymerized at 60 ◦C for2 days. ∼80 nm-thick sections were collected on 75-mesh cop-per grids, counterstained with uranyl acetate and lead citrate andobserved in a JEOL 1010 TEM operating at 80 kV. For the study ofthe different structures present in the interchromatin region, themethylation–acetylation (MA) and the EDTA cytochemical meth-ods were used, either separately or combined. For the preferentialstaining of DNA, the NAMA-Ur (Testillano et al., 1991) ultrastruc-tural cytochemistry was used. The MA method is a pre-embeddingtechnique that highlights nucleic acid-containing structures (fib-rillar and granular) by a selective blockage of the protein ability toreact with uranyl acetate (Testillano et al., 1995). For this method,fixed samples were dehydrated in a methanol series and incu-bated in a freshly made methanol:acetic anhydride mixture (5:1v:v) overnight at room temperature, prior to embedding. Afterthree washings with methanol, samples were embedded in Epon

as described. For the NAMA-Ur cytochemical method, fixed sam-ples were subjected to a mild alkaline hydrolysis with NaOH toeliminate RNA, followed by MA treatment and Epon embedding;subsequent uranyl staining of the sections result in a preferentialstaining of DNA-containing structures (Testillano et al., 1991). The

748 J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757

F nd yoM MDEs

Etoiiem

2i

odsgi2lTE−sf2maaipm

2

fii(mLAbdiBwa

ig. 1. Early stages of microspore embryogenesis. (A) Freshly isolated microspores aDE. (E) Heart-shaped MDE. Insets in figures A–D are sections of microspores and

DTA regressive staining (Bernhard, 1969) is used over EM sec-ions to provide RNP-containing structures a preferential stainingver the bleached masses of condensed chromatin. Samples fixedn 4% formaldehyde, dehydrated by the PLT (Progressive Lower-ng of Temperature) method in a Leica AFS automatic system, andmbedded in Lowicryl K4 M resin were used for this cytochemicalethod.

.3. Cryoprocessing of samples for cryomicrotomy andmmunofluorescence

Microspore and microspore-derived embryo cultures were cry-processed for cryomicrotomy and cryosectioned as previouslyescribed (Seguí-Simarro et al., 2003). Microspore cultures werelightly prefixed in 4% formaldehyde at 4 ◦C and embedded inelatin. Small (1 mm3) pieces of gelatin were cryoprotected withncreasing concentrations of sucrose: 0.1 M (1 h), 1 M (3 h), and.3 M (overnight), placed on an aluminum pin and cryofixed in

iquid propane using a KF80 unit (Reichert, Vienna) at −170 ◦C.hin (1 �m thick) cryosections were obtained using an Ultracut(Reichert) ultramicrotome coupled with a FC4 unit stabilized at75 ◦C. Cryosections were collected and placed on multiwell glass

lides for immunofluorescence. Immunofluorescence was also per-ormed essentially as described previously (Seguí-Simarro et al.,003), with the primary mouse monoclonal antibody anti-2,2,7-tri-ethyl-guanosine, TMG (Calbiochem, clone K121) diluted 1/100

nd incubated for 1 h at room temperature, and the secondaryntibody (anti-mouse IgG-Cy3) diluted 1:25 in 1% BSA in PBS, andncubated for 45 min. Sections were additionally stained with DAPIrior to observation. Controls were performed excluding the pri-ary antibody.

.4. Immunoelectron microscopy

Microspores and haploid embryos at different stages were pre-xed in formaldehyde, cryoprotected in 2.3 M sucrose, cryofixed

n liquid nitrogen, and cryoprocessed as described previouslySeguí-Simarro et al., 2003). Samples were freeze-substituted in

ethanol + 0.5% uranyl acetate at −80 ◦C for 3 days, infiltrated inowicryl K4 M, and polymerized at −30 ◦C under UV light in a LeicaFS system. Ultrathin (80 nm) sections were placed on nickel grids,locked with 5% BSA in PBS, and incubated with anti 2,2,7-TMG

iluted 1/100 for 1 h at room temperature. Then, the grids were

ncubated with a secondary antibody (anti-mouse IgG-gold, 10 nm;iocell) diluted 1:25 in 1% BSA, for 45 min at room temperature,ashed, air dried, counterstained, and observed in a JEOL 1010 EM

t 80 kV. Controls were performed excluding the primary antibody.

ung bicellular pollen. (B) Induced microspores. (C) Early (octant) MDE. (D) Globularat the corresponding stages. Bars in A, B: 10 �m. C–E: 50 �m.

2.5. Morphometric and quantitative analysis

Sampling was carried out over selected samples on eachgrid. The number of micrographs to be taken was determinedby the progressive mean test (Williams, 1977), with a maxi-mum confidence limit of a = 0.05. Digital images of cells showingone or more CBs were processed with the ImageJ software(http://rsbweb.nih.gov/ij/) for the morphometric analysis of num-bers, areas, and distances. Data were exported to a spreadsheet,where the final calculations and chart displays were performed.

3. Results

The first step of the study was the selection of the most represen-tative stages of microspore embryogenesis and pollen developmentto be processed and analyzed. For microspore-derived embryoge-nesis, we chose the earliest stages, immediately before and afterembryogenesis induction, where the most dramatic rearrange-ments at the structural, ultrastructural and molecular levels havebeen described (Maraschin et al., 2005; Seguí-Simarro and Nuez,2008). These stages comprise: (1) vacuolate microspores and youngbicellular pollen just extracted from the anther but prior to expo-sure to the inductive heat shock (Figs. 1A and 2A) and (2) justafter induction (Fig. 1B), where the first sporophytic divisions areseen still within the exine coat (inset in Fig. 1B); (3) induced,early microspore-derived embryos (MDEs; Fig. 1C), once releasedfrom the exine coat and with two clearly differentiated suspensorand embryo proper domains; (4) globular MDEs (Fig. 1D), wherethe first signs of tissue differentiation are visible in the embryoproper domain (inset in Fig. 1D); and (5) heart-shaped MDEs(Figs. 1E and 2B), where the embryo pattern of symmetry changesfrom radial to bilateral, hypocotyl and cotyledon begin to elongateand differentiation of internal tissues takes place (Tykarska, 1980).For pollen development, we selected (1) the vacuolate microspore,before the first pollen mitosis, (2) the young pollen grain (Fig. 3A)immediately after the first pollen mitosis, (3) the mid pollen grain(Fig. 3B), when the generative cell displaces towards the center ofthe vegetative cell, and (4) the late, mature pollen grain (Fig. 3C),when sperm cells are formed after the second pollen mitosis, stillwithin the anther locule.

3.1. Nuclear size, chromatin condensation pattern and nucleolar

architecture

The nucleus of vacuolate microspores was typically round orslightly oval, large and off-center (Figs. 1A inset, 2A) due to thepresence of a large central vacuole. Chromatin displayed the decon-

J.M. Seguí-Simarro et al. / Journal of Plan

Fig. 2. Overview of the nuclear architecture during microspore embryogenesis. Vac-uolate microspore (A) and heart-shaped MDE nuclei (B) in MA-treated sections. Notethe remarkably different organization of the different components of the interchro-matic region (ir) in both cell types. cb: Cajal body; chr: condensed chromatin; dfc:nnv

dotcdadndtcmcmcsa

oaat

preferential for DNA (Testillano et al., 1991), clearly revealed

ucleolar dense fibrillar component; ex: exine coat; fc: nucleolar fibrillar center; gc:ucleolar granular component; ne: nuclear envelope; nu: nucleolus; nv: nucleolaracuole; v: vacuole. Bars: 500 nm.

ensed pattern typical of this species, with only few, large massesf condensed chromatin, often associated with the inner side ofhe nuclear envelope (Fig. 2A), allowing for an abundant inter-hromatin region. In induced microspores, the first embryogenicivision was symmetric and gave rise to two similar nuclei, equiv-lent in size (Fig. 6C), rounded and centrally positioned. Subsequentivisions of the embryo proper cells produced progressively smalleruclei (Fig. 4A). This nuclear morphology was observed, with slightifferences, in interphasic nuclei of microspores and MDEs throughhe early stages of embryogenesis. The chromatin pattern in theseells could be defined as dark, electron dense masses in electronicroscopy sections of samples treated with the nucleic acid-

ontrasting MA cytochemistry (Fig. 2A and B), as clear, bleachedasses (Fig. 5A) in sections treated with the EDTA regressive cyto-

hemical staining, or as intense, peripheral bright spots in nucleitained with the fluorescent, DNA-specific DAPI staining (Fig. 6And D).

This pattern strikingly contrasted with that observed in devel-

ping pollen under non-inductive conditions (Fig. 3). Immediatelyfter the first pollen mitosis (Fig. 3A), a large, vegetative (VN) andsmaller, generative nucleus (GN) appeared within the vegeta-

ive and generative cells. As pollen maturation proceeded, the VN

t Physiology 168 (2011) 746–757 749

retained an architecture comparable to that described for vacuo-late microspore nuclei (compare Figs. 3B–B’ and 2A). However,as the generative cell moved from the periphery to the centerof the vegetative cell, the size of the GN dramatically decreased(Figs. 3B and 4B) and chromatin adopted a highly condensed status,appearing as large bleached masses after EDTA staining (Fig. 3B”).In the mature pollen grain, the VN showed signs of increased chro-matin condensation (Fig. 3C’), while the nuclear envelope adoptedirregular, lobed morphologies (Fig. 3C). After the second pollenmitosis, the two newly formed sperm nuclei occupied nearly allof the volume of the sperm cells (Fig. 3C). Chromatin condensa-tion reached the highest levels, appearing in MA-treated samplesas large, densely stained masses (Fig. 3C) and as bleached areas inEDTA samples (Fig. 3C”).

It is well established that changes in the transcriptional sta-tus of the cell are accompanied by significant extensive changesin nucleolar volume and morphology (Raska et al., 1983; Goessens,1984). Thus, we used the nucleolar organization as a reliable markerto identify cells at different transcriptional status. At the vacuo-late microspore stage, the nucleolus was round, centered, largewith respect to the total nuclear volume (Fig. 2A), and displayinga torus-shaped profile typical of highly active cells: a large cen-tral vacuole with ribonucleoprotein particles (RNPs), and abundantgranular component (GC) intermingled with dense fibrillar com-ponent (DFC). In the DCF, many small and homogeneous fibrillarcenters (FCs) were observed (Fig. 2A). In MDEs, the nucleolar archi-tecture appeared more variable. Indeed, besides the torus-shapedprofiles indicative of highly active nucleoli, profiles indicative ofmoderate and low activity were also observed in other cells. Profilesof moderate activity were defined by the presence of many small,homogeneous FCs within a DFC with abundant GC (data not shown).Low activity profiles included few, large and heterogeneous FCs,and scarce GC, usually located at the periphery of the DFC (Fig. 2B).During pollen development, the nucleolus of the generative cellfrequently showed low activity profiles just after the first pollenmitosis (Fig. 3A), whereas profiles of moderate activity were evi-dent in the vegetative nucleus up to the mid pollen stage (Fig. 3B).In mature pollen grains, the nucleolus was reduced to a compact,dense and inactive structure in the generative nucleus and in spermcells (Fig. 3C). In the vegetative nucleus, nucleoli were visible up tothe mid pollen stage.

3.2. Cytochemical analysis of the interchromatin region

In MA-treated samples, condensed chromatin masses showedhigher contrast than the nucleolus and interchromatin RNP struc-tures (Fig. 5A), with the chromatin patches clearly defined. EDTAnuclear staining revealed some interesting and novel features ofthe plant IR. At a first visual inspection, the areas of EDTA-bleached,condensed chromatin masses (Fig. 5B) were larger than their equiv-alents in MA (non-EDTA)-treated samples (Fig. 5A). To verify thisobservation, we quantitatively evaluated the areas of large chro-matin masses in 100 randomly chosen MDE cells from EDTA andMA-treated sections. We found that the average area of a bleachedchromatin region in EDTA-stained samples was 0.47 ± 0.29 �m2,whereas in MA-stained samples it was 0.37 ± 0.23 �m2. Thisdifference (∼21%) indicated that EDTA bleaches not only chro-matin masses, but also the adjacent, perichromatin region whereabundant chromatin fibers of different thickness are probably pref-erentially located. The use of the NAMA-Ur cytochemical method,

stained numerous chromatin fibers of different thicknesses at theperiphery of the chromatin masses (Fig. 5C, arrows). The compari-son of these cytochemical techniques suggested the existence of aperichromatin region, rich in decondensed chromatin.

750 J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757

F ries op and Ac -treate

RmwweiitEttB

aid(una

ig. 3. Changes in nuclear architecture during pollen maturation. The A, B and C seollen, mid bicellular pollen and mature, tricellular pollen, respectively. The A’B’C’orrespond to a sperm nucleus of tricellular pollen. All figures but C are from EDTA

EDTA also revealed the presence of a dense fibrillo-granularNP network filling the nucleoplasmic space between chromatinasses. In all of the embryogenic and gametophytic stages studied,e observed the presence of perichromatin fibrils (9.9 ± 1.24 nmide), either emerging from the mass borders or as isolated threads

mbedded in the IR (Fig. 6B). Isolated granules, 37.8 ± 5.96 nmn diameter, were observed dispersed through the IR, mostly innduced microspores and MDEs. They were frequently seen athe nuclear periphery, close to the nuclear envelope (Fig. 6D and). Their size, spherical shape, staining properties and principallyhe presence of a defined hallo around them, allowed us to iden-ify them as perichromatin granules according to (Monneron andernhard, 1969).

Smaller (22.3 ± 5.53 nm in diameter) spherical granules werelso found in the IR (Fig. 6A and C), both isolated and groupednto clusters over a fibrillar matrix (Fig. 5C). According to their

istribution, these granules were likely interchromatin granulesIG). Correspondingly, clustered IGs would be interchromatin gran-le clusters (IGCs), the electron microscopy equivalents of theuclear speckles observed by fluorescence microscopy (Lamondnd Spector, 2003). In some sections, we were able to iden-

f figures cover the changes in nuclear organization undergone by young bicellular”B” columns show the IR of the vegetative and generative nucleus respectively. C”d sections. See text for further details. Bars in A–C: 5 �m. A’–C”: 500 nm.

tify a clearer, mostly fibrillar region devoid of IGs but alwaysadjacent to IG clusters (Fig. 6C, dotted lines). The structureof these regions highly resembled the “interchromatin granule-associated zone” previously described in mammalian cells (Visaet al., 1993).

IGCs were evident throughout microspore embryogenesis, firstin dividing microspores, and to a lesser extent in MDEs. Duringpollen development, IGCs were observed soon after the first pollenmitosis (Fig. 3A–A”). However, their presence was progressivelyreduced during pollen maturation. Isolated IGs were observed asdark, well defined, isolated particles through the progressivelyreduced IR of the early (Fig. 3A–A”), mid (Fig. 3B–B”) and finallymature pollen grain (Fig. 3C–C”). This was paralleled by a pro-gressive decrease in nuclear size and a remarkable increase in thecondensation status of chromatin. The intensity of the transforma-tions was higher in the generative nuclei, GNs (compare Fig. 3B–B”),

and in sperm cells (Fig. 3C”), and with less intensity in the vegeta-tive nuclei, VNs. In summary, these results indicated a relationshipbetween the increase in chromatin condensation, the IR reductionand a change in the distribution pattern of IGs from clustered toisolated during pollen maturation.

J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757 751

F rea dua sporeh ther d

3

i((eeoaTieotpfttdsf

tt(

FNRNt

ig. 4. Morphometric analysis of nuclear size. Estimation by the average nuclear average nuclear area over TEM micrographs measured in �m2. Mic: isolated microeart-shaped MDE; Nv: vegetative nucleus; Ng: generative nucleus. See text for fur

.3. Nuclear immunolabeling with anti 2,2,7-trimethylguanosine

Further characterization of the IR substructures involvedmmunolabeling with antibodies against 2,2,7-trimethylguanosineTMG), the specific cap present in the RNA of the small nuclear RNPssnRNPs). SnRNPs are major components of the splicing machin-ry (Lührmann, 1988). Anti-TMG signal was observed in all of thembryogenic stages studied during MDE development. Inmunoflu-rescence signal (Fig. 7) was located at the nucleus in the form offaint signal and some discrete, round and intensely bright foci.

hese foci never colocalized with those observed for DAPI stain-ng (compare Fig. 7A–E and A’–E’), indicating that chromatin isxcluded from them. The nucleolus always appeared devoid of flu-rescence as well, indicating that these structures were confined tohe IR and most probably corresponded to nuclear bodies. Duringollen maturation, the faint fluorescence was negligible, and veryew foci could be observed after the first pollen mitosis, mainly inhe VN (Fig. 7B’). As the chromatin of the pollen nuclei condensed,he TMG-labeled structures were less evident, becoming no longeretectable in mature pollen (Fig. 3C). Therefore, we focused on thetudy of these structures during MDE development, where intenseoci were present (Fig. 7C–E’).

Anti-TMG immunogold labeling confirmed a faint labelinghroughout different regions of the IR. At the boundaries betweenhe chromatin masses and the IR, significant labeling was foundFig. 8C, arrowheads), possibly detecting snRNPs associated to

ig. 5. The condensed chromatin and the perichromatin region after cytochemical technuclear regions showing a chromatin mass. (A) MA staining for nucleic acids; condensed cNP structures. (B) EDTA staining for RNPs; condensed chromatin (chr) appears bleachedAMA-Ur staining for DNA; only condensed chromatin masses (chr) and chromatin fibe

he nucleolus (nu) and cytoplasm (ct) show no contrast at all. Bars: 300 nm.

ring microspore embryogenesis (A) and pollen maturation (B). Bars represent thes; Ind: induced microspores; EG: early globular MDE; LG: late globular MDE; HS:etails.

nascent transcripts. The nucleolus was nearly devoid of labeling.Labeling of the IR appeared either as isolated particles or as clus-ters of few (2–6) particles (Fig. 8A–C). By combining immunogoldlabeling with EDTA staining it was shown that particle clusters dec-orated EDTA-positive, RNP-containing IR regions (Fig. 8D, arrows),in a pattern similar to that described for IGCs. Thus, the clusters ofgold particles of the IR would actually correspond to the granularIGC regions observed in Epon-embedded samples, although with adifferent appearance, due to the different processing, as describedpreviously in other studies of the IR (Testillano et al., 1993).

In addition to small clusters, immunogold particles concen-trated strongly over dense, coiled-looking bodies (Fig. 8A–E) thatcorresponded to the bright foci observed with immunofluores-cence according to their size and position. These bodies frequentlyexhibited a nearly circular section, variable in size, and a thickfibrillar texture embedded into a light matrix continuous withthe IR. Immunogold labeling decorated RNP-positive, particle-likestructures contained within the matrix of the body (Fig. 8D). Thestructure, size, nature and position of these bodies, together withthe labeling with anti-TMG, indicative of the presence of RNPs, arecharacteristics of Cajal Bodies (CBs; (Fakan et al., 1984; Spectoret al., 1991; Spector, 1993a; Seguí-Simarro et al., 2006). In addi-

tion, where the sectional plane was appropriate, we also observeda clearer, homogeneous and scarcely labeled region associated withthe CB (cb–az in Fig. 8E) and structurally different from other sub-nuclear structures. The structural characteristics of these novel

iques for nucleic acids (MA), RNPs (EDTA) and DNA (NAMA-Ur) in globular MDEs.hromatin (chr) displays higher contrast than the nucleolus (nu) and interchromatinwhereas the nucleolus (nu) and RNP fibers and granules of the IR show contrast. (C)rs at the perichromatin region (arrows) and in the IR show high contrast, whereas

752 J.M. Seguí-Simarro et al. / Journal of Plan

Fig. 6. The interchromatin region in EDTA-treated sections of globular MDE cells.(A) nuclear overview showing the nucleolus (nu), the bleached chromatin masses(chr) and the interchromatin region (ir). (B, C) magnifications of the regions boxedigmn

rb1

3

omctutgm

bcdio

n A. Arrowheads in B: perichromatin fibrils. Dotted line in C: interchromatinranule-associated zone. D: interchromatin region with perichromatin granules. E:agnification of the area boxed in D. Arrows point to perichromatin granules. ne:

uclear envelope. Bars in A: 500 nm. B–E: 200 nm.

egions, not described to date in plants, resembled the cleavageodies described as associated with CBs in animal cells (Schul et al.,996; Gall, 2000).

.4. Qualitative and quantitative analysis of Cajal bodies

According to the criteria detailed above, we identified CBs in allf the embryogenic stages studied. Interestingly, in freshly isolatedicrospores we noted a relatively lower electron density of CBs

ompared to CBs of other stages (compare Fig. 8A and B). Most ofhe CBs in just-induced microspores (not shown) and early glob-lar MDEs (Fig. 8B) presented a similar appearance, in general, inerms of electron density (high) and circularity (reduced). In latelobular and heart-shaped MDEs, circular, electron light CBs wereore abundant.For the quantitative analysis of CBs, we first estimated the num-

er (Fig. 9A) and area (Fig. 9B) of CBs at each stage in order to have alear view of their abundance during MDE development. Given theifferences in nuclear size at different stages (Fig. 2C), we normal-

zed the nuclear density and size of CBs expressing them as numberr area of CBs per nuclear �m2. Fig. 9A shows that microspores

t Physiology 168 (2011) 746–757

prior to induction presented comparatively less CBs than any otherstage. Commitment of microspores to embryogenesis was paral-leled by an increase in the number of CBs, peaking in early globularMDEs. In late globular and heart-shaped MDEs, when tissue dif-ferentiation starts, the number of CBs showed a similar, reducedvalue, slightly higher than in microspores. A completely oppositetrend was reflected in mean CB area calculations (Fig. 9B). Afterinduction, the size of CBs was drastically reduced, increasing pro-gressively through the following stages of embryogenesis. Thus,an inverse relationship between CB size and number was evident(compare the trend lines of Fig. 9A and B). After measuring the totalarea occupied by CBs at each stage (Fig. 9C), we confirmed this rela-tionship, finding that the total area was nearly constant except forone stage. In induced microspores, the total CB area was markedlylower.

An additional unexpected finding arose when we compared theerror bars of the different stages. Since the measured area of anearly spherical object like a CB depends on the sectional plane, onewould expect an error proportional to the measured area, and sim-ilar for all of the stages. However, in freshly isolated microspores,the error was comparatively smaller than in the other stages, sug-gesting that CBs in this stage are larger and more uniform in size.To investigate this, we analyzed the frequency distribution of theareas of all of the CBs from the different stages considered alto-gether (Fig. 9D). We found that areas did not exhibit a gaussiandistribution, as expected for a uniform size. Instead, they showeda bimodal pattern, with measurements congregating around twopeaks at 0.15 and 0.35 �m2, which would correspond to estimateddiameters of 0.44 and 0.67 �m, respectively. Thus, it appeared thatCBs may have two different sizes, large and small, which accordingto our micrograph analysis correspond to the electron light (Fig. 9E)and electron dense (Fig. 9F) types described above, respectively.CBs of freshly isolated microspores would belong almost exclu-sively to the large and electron light type, whereas after induction,both types may coexist, with the small more frequent in early post-inductive stages and the large more frequent in later embryogenicstages. Once two different CB categories were established, we reex-amined the presence of the CB-associated zones, and interestingly,they were exclusively found to be associated with CBs of the smalland dense type. In summary, it appeared that CBs change theirsize, number, shape, and position in a defined manner during earlyMDE development, whereas they disappear during pollen matura-tion. These data would indicate that the described changes are areflection of the change in developmental program.

4. Discussion

The comparison between the gametophytic and sporophyticpathways followed by the microspore permitted us to analyze theremodeling of the nuclear domains in plant differentiating cellswhen switched to proliferation. The change of the developmen-tal program and the activation of the proliferative activity affectedthe functional organization of the interchromatin domain, whichchanged the architecture and functional state, and redistributed itscomponents. A summary of the main nuclear changes observed isshown in Fig. 10.

In this work, we present an ultrastructural, cytochemical andimmunocytochemical characterization of the changes in nucleararchitecture, with special attention to the IR-housed structures,during two different developmental processes with the same

starting point: differentiation of microspores into mature, gamete-harboring pollen grains, transcriptionally inactive (Bednarska,1984), and proliferation of embryogenesis-induced microsporesduring the first embryogenic stages, highly engaged in transcrip-tion (Joosen et al., 2007; Malik et al., 2008). The comparison of the

J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757 753

F embp imago

ccdip

4d

n

ig. 7. Anti-TMG immunofluorescence over cryosections during gametophytic andoint to CBs. Note the absence of DNA in the corresponding regions in DAPI-stainedf the microspore. Bars: 10 �m.

hanges undergone by the structures of the IR during these two pro-esses allowed us (1) to relate the remodeling of the interchromatinomain to the change of developmental program and to variations

n transcriptional activities, and (2) to reveal novel features of thelant nucleus, not yet described.

.1. Changes in chromatin pattern parallel the change ofevelopmental program

Numerous studies have shown a direct relationship betweenuclear architecture and the level of cellular activity (Risueno and

ryogenic development. (A–E) DAPI staining. (A’–E’) Anti-TMG. Yellow arrowheadses (compare white arrows in A and A’). The asterisk in A marks the massive vacuole

Testillano, 1994; González-Melendi et al., 1998; Testillano et al.,2000, 2005; Raska et al., 2004). In this work, we showed that thechromatin pattern is also affected by the induction of microsporeembryogenesis in B. napus, constituting a good marker of develop-mental fate. In B. napus microspores committed to embryogenesis,all nuclei show a similar decondensed chromatin pattern, allowing

for an abundant IR. The scarce chromatin masses are mostly asso-ciated with the nuclear envelope. This pattern, similar to that ofsomatic cells, could be considered a variation of the chromomericcondensation pattern (Jordan et al., 1980). This pattern changes inpollen cells, where a strikingly different, highly condensed chro-

754 J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757

Fig. 8. Anti-TMG immunogold labeling in microspores and MDEs. (A) Interchromatin region (ir) of a vacuolate microspore showing an intensely labeled Cajal body (cb).(B) Interchromatin region of an early globular MDE showing abundant labeling over one electron-light (centrally located) and two electron-dense CBs (at both sides). (C)O nd anA articlC n EDT(

mtcic

4e

sidimtlD

verview of the interchromatin region of a heart-shaped MDE, showing a large, rourrows indicate clusters of gold particles over the IR, whereas arrowheads point to pB-marking gold particles concentrate into small clusters on the IR (arrows), over acb–az). ct: cytoplasm; ex: exine coat; ne: nuclear envelope. Bars: 200 nm.

atin pattern is observed. The embryogenic switch originates inhe microspore and MDEs a characteristic organization with lessondensed chromatin pattern the chromatin, typical of proliferat-ng cells, and markedly different from developing microspores notommitted to embryogenesis.

.2. Changes in IR structures associated to induction ofmbryogenesis

In this work, we show evidence of a remodeling of the IR-housedtructures as a consequence of the developmental switch of thenduced microspore. IGs change from grouping as IGCs mostly inividing microspores (transcriptionally active) to being isolated

n maturing pollen and gametes (transcriptionally inactive). Inammalian cells, various reports have shown that a transcrip-

ion blockage resulted in the accumulation of splicing factors inarger speckles/IGCs (Spector et al., 1983, 1991; Melcak et al., 2000;ocquier et al., 2004). Nevertheless, in plant cells, the organiza-

d circular CB between the nucleolus (nu) and a mass of condensed chromatin (chr).es decorating the borders of chromatin masses. (D, E) EDTA-treated sections whereA-positive (RNP-containing) CB (outlined in D), and also over a CB-associated zone

tion of the IGs differs slightly. IGs have been observed as isolatedgranules in many plant cell systems (Testillano et al., 1993), asthe splicing factors distribution was more spread throughout theIR, as revealed by immunofluorescence assays. In the generativeand sperm cells, the nuclear size progressively decreases as thechromatin adopts an extremely condensed pattern, which dramat-ically reduces the space available for the IR to a minimal amount(Fig. 3B” and C”). This lack of space may be affecting IGC nucleation.In Arabidopsis trichome cells, with extremely high endoredupli-cation levels, a transcription blockage causes the appearance ofthousands of microspeckles, instead of the expected larger speck-les (Fang et al., 2004). In parallel, the inconspicuous presence ofIGCs in dividing microspores could be related to the application of

a heat shock treatment to induce the developmental switch. Indeed,heat shock has been shown to promote the accumulation of plantsplicing factors in larger speckles/IGCs (Docquier et al., 2004).

Other features of the IR associated with the change of devel-opmental program affected PGs and PFs. The increased number

J.M. Seguí-Simarro et al. / Journal of Plan

Fig. 9. Quantitative analysis of Cajal bodies during microspore embryogenesis. (A)mean CB number per nuclear �m2. (B) Mean individual CB area per nuclear �m2.(C) Mean total CB area per nuclear �m2. (D) Frequency distribution, expressed inpercentage, of the areas of the different CBs observed. See text for further details. (E)example of a large, electron light CB. (F) example of small, electron dense CB. Mic:isolated microspores; Ind: induced microspores; EG: early globular MDE; LG: lateglobular MDE; HS: heart-shaped MDE. Bars: 200 nm.

t Physiology 168 (2011) 746–757 755

of PGs observed after the embryogenesis-inductive heat-shockwould support a similar role for plant PGs on the accumulationof heat shock-damaged pre-mRNA molecules than in mammaliancells (Puvion and Lange, 1980). In HeLa cells, Chiodi et al. (2000)showed the formation of PG clusters upon heat shock exposure.However, we could not identify such a clustered pattern in anyof our plant samples. EDTA staining revealed a perichromatinregion of decondensed chromatin, rich in decondensed chromatin,which would correspond to active genes ready for transcriptionand/or replication. The RNP fibrils (PFs) were further away from thechromatin masses, and would correspond to the different stagesof hnRNA transcription and processing. Moreover, we identifieda distinct region of ribonucleoproteic nature, structurally simi-larly to the IG-associated zone (IG-AZ) of HeLa cells (Visa et al.,1993). The IG-AZ is considered as a part of the nuclear matrixthat contains p80-coilin and U1 snRNA (but not U2), and is thusthought to be a reservoir of spliceosome components (Puvion-Dutilleul et al., 1995). These zones have never been described inplants before, and their presence in certain cells could be indicat-ing higher transcriptional activity. Nevertheless, further structuraland immunochemical studies would be necessary to characterizethe functional role of this nuclear region in plants.

4.3. Novel features of plant Cajal bodies

The combination of anti-TMG immunolabeling with ultrastruc-tural cytochemistry allowed us to identify several novel featuresof the plant CB. One is the identification of two ultrastructurallydifferent types of CBs in B. napus. Despite of their differences,our structural and quantitative data reveal a number of similari-ties between them, highly suggestive of a similar nature for both.Indeed, they may actually be the divided and fused forms of thesame subnuclear structure, as it is known that CBs are highlydynamic structures, undergoing frequent movements from/to thenucleolus, and also fission and fusion between them (Lafarga et al.,1998; Boudonck et al., 1999; Platani et al., 2000; Navascués et al.,2004). This plasticity may be the origin of the heterogeneity in CBstructure found in the B. napus nucleus as well as in other plant celltypes such as maize root cells (Docquier et al., 2004), heterogeneitythat is also observed in some animal cells (Liu et al., 2006).

We identified a clearer, large CB-associated zone (Fig. 7E) alwayscontacting CBs of the small, dense type. These regions are struc-turally similar to those described in animal cells as CB-associatedcleavage bodies due to the presence of, among others, factors forthe cleavage of the 3′-end of polyadenilated mRNAs (Schul et al.,1996; Gall, 2000). A role for cleavage bodies related to the pro-cessing of specific pre-mRNA subsets not found in their associatedCBs has been proposed (Schul et al., 1996). Since the functionalrole of CBs is similar in plant and animal cells, it is likely thatthese plant CB-associated zones could have a similar role as well,although more studies combining structural characterization within situ molecular localization are needed to further characterize thisnovel compartment. Ultrastructural studies have found CBs and IGsassociated in proliferating plant cells (Moreno Díaz de la Espinaet al., 1982); since CBs are mobile bodies and we have found sim-ilar AZs associated to IGs, the possibility that the three structures,CBs, IGs and AZ were associated cannot be excluded, although it isvery difficult to find the three in the same section.

It is known that the number of CBs in a cell is developmen-tally and cell cycle-regulated (reviewed in Shaw and Brown, 2004),cell type-specific (Raska et al., 1991) and tightly related to the

changes in transcriptional and metabolic activity (Carmo-Fonsecaet al., 1992, 1993; Spector, 1993b). Our results showed that thenumber and size of CBs are tightly related to proliferation, in adirect manner for number, and inversely for size. The opposite sce-nario would apply for the relationship between CB size and number

756 J.M. Seguí-Simarro et al. / Journal of Plant Physiology 168 (2011) 746–757

F in boe ies. −

dohttece

is2vttoamt

4

iioisrrm

lcbstaslto2

ig. 10. Summary of the changes in the chromatin condensation and IR structuresmbryogenesis (proliferation process). IGs: interchromatin granules, CBs: Cajal bod

uring differentiation events. The only exception to this trend wasbserved in induced microspores exiting from a recently appliedeat shock, where clear deviations were observed in individual andotal CB size (Fig. 9B and C). Indeed, exposure to heat shock induceshe formation of smaller CBs in different animal cells (Handwergert al., 2002). Similar evidences have also been observed in HeLaells, where exposure to high temperatures produced smaller CBs,ven losing their contents in snRNPs.

A model has been proposed in sugarcane root primordial cellsn which the number of CBs per nucleus would be fixed for a givenpecies and cell type, independent of its activity (Acevedo et al.,002). Our results would be in agreement with this notion, pro-ided that what remains constant is the total “mass” of CBs, not onlyheir number, and under non-stressing conditions. Taken together,hese results would indicate that the total CB “mass” within a nucle-plasm remains nearly constant during MDE development in thebsence of stress. Upon stressing the cell, CBs would fragment intoini-CBs and dismantle, but in a reversible manner, recovering the

otal CB “mass” to normal levels after stress.

.4. Concluding remarks

In this work, we have shown that the different structuresnvolved in RNA processing change their shape and distributionn a characteristic manner as a consequence of the reprogrammingf the microspore from the natural, gamete-producing route to the

n vitro, embryo-producing route. Some features of the IR were heathock-derived consequences. After heat shock, CBs considerablyeduce their individual size and their total volume, although in aeversible manner. The increased occurrence of IGCs in inducedicrospores could also be a heat shock effect.Other IR changes were associated with the commitment to pro-

iferation (in MDEs) or to differentiation (in maturing pollen). Thehange of IR volume, the distribution pattern of IGs, and the num-er and size of CBs were related to a change in the transcriptionaltatus of the cell. Moreover, novel features of plant IR-housed struc-ures were found, such as the presence of two IR zones specificallyssociated to IGCs and CBs, the existence of heterogeneity in the

tructure of the plant CB, with a smaller and denser type, and aarger and lighter type. A significant remodeling of the microsporeranscriptome has been recently described as an essential partf the microspore switch to embryogenesis (Hosp et al., 2007a,007b; Joosen et al., 2007; Malik et al., 2007). While chromosome

th pollen programs: pollen development (differentiation process) and microspore: absence, +/−: low amount, +: present, ++: abundant, +++: high abundant.

remodeling during reprogramming (Hosp et al., 2007a) would actas a pre-translational mechanism for regulation of gene expres-sion, the changes described in the present work of the structuresassociated to RNA processing machinery would serve as a first post-translational regulatory mechanism.

Acknowledgements

This work was supported by grants from the Spanish Ministryof Education and Science AGL2006-06678 to J.M.S.S., AGL2008-04255 to M.C.R. BFU2008-00203 to P.S.T. and Czech grantsMSM0021620806, LC535 and AV0Z50110509 to I.R.

References

Acevedo R, Samaniego R, de la Espina SMD. Coiled bodies in nuclei from plant cellsevolving from dormancy to proliferation. Chromosoma 2002;110:559–69.

Andrade LE, Tan EM, Chan EKL. immunocytochemical analysis of the coiled body inthe cell cycle and during proliferation. Proc Natl Acad Sci USA 1993;90:1951.

Barany I, Fadon B, Risueno MC, Testillano PS. Cell wall components and pectin ester-ification levels as markers of proliferation and differentiation events duringpollen development and pollen embryogenesis in Capsicum annuum L. J ExpBot 2010;61:1159–75.

Bednarska E. Ultrastructural and metabolic transformations of differentiatingHyacinthus orientalis L. pollen grain cells. I. RNA and protein synthesis. ActaSocietatis Botanicorum Poloniae 1984;53:145–58.

Bernhard W. A new staining procedure for electron microscopical cytology. J Cell Sci1969;27:250–65.

Boudonck K, Dolan L, Shaw PJ. Coiled body numbers in the Arabidopsis root epider-mis are regulated by cell type, developmental stage and cell cycle parameters. JCell Sci 1998;111:3687–94.

Boudonck K, Dolan L, Shaw PJ. The movement of coiled bodies visualized in livingplant cells by the green fluorescent protein. Mol Biol Cell 1999;10:2297–307.

Carmo-Fonseca M, Ferreira J, Lamond AI. Assembly of snRNP-containing coiled bod-ies is regulated in interphase and mitosis – evidence that the coiled body is akinetic nuclear structure. J Cell Biol 1993;120:841–52.

Carmo-Fonseca M, Pepperkok R, Carvalho MT, Lamond AI. Transcription-dependentcolocalization of the U1, U2, U4/U6 and U5 snRNPs in coiled bodies. J Cell Biol1992;117:14.

Cmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu XD, et al. Ultrastruc-tural analysis of transcription and splicing in the cell nucleus after bromo-UTPmicroinjection. Mol Biol Cell 1999;10:211–23.

Cremer T, Cremer C. Chromosome territories, nuclear architecture and gene regula-tion in mammalian cells. Nat Rev Genet 2001;2:292–301.

Chiodi I, Biggiogera M, Denegri M, Corioni M, Weighardt F, Cobianchi F, et al.Structure and dynamics of hnRNP-labelled nuclear bodies induced by stresstreatments. J Cell Sci 2000;113:4043–53.

Docquier S, Tillemans V, Deltour R, Motte P. Nuclear bodies and compart-mentalization of pre-mRNA splicing factors in higher plants. Chromosoma2004;112:255–66.

f Plan

F

F

F

GGG

H

H

H

J

J

J

L

L

L

L

L

M

M

M

MM

M

M

N

P

P

P

P

J.M. Seguí-Simarro et al. / Journal o

akan S. Ultrastructural cytochemical analyses of nuclear functional architecture.Eur J Histochem 2004;48:5–14.

akan S, Leser G, Martin TE. Ultrastructural distribution of nuclear ribonucleo-proteins as visualized by immunocytochemistry of thin sections. J Cell Biol1984;98:358–63.

ang YD, Hearn S, Spector DL. Tissue-specific expression and dynamic organizationof SR splicing factors in Arabidopsis. Mol Biol Cell 2004;15:2664–73.

all JG. Cajal bodies: the first 100 years. Annu Rev Cell Dev Biol 2000;16:273–300.oessens G. Nucleolar structure. Int Rev Cytol 1984;87:107–58.onzález-Melendi P, Testillano PS, Mena CG, Muller S, Raska I, Risueno MC. His-

tones and DNA ultrastructural distribution in plant cell nucleus: a combinationof immunogold and cytochemical methods. Exp Cell Res 1998;242:45–59.

andwerger KE, Wu ZA, Murphy C, Gall JG. Heat shock induces mini-Cajal bodies inthe Xenopus germinal vesicle. J Cell Sci 2002;115:2011–20.

osp J, Maraschin SDF, Touraev A, Boutilier K. Functional genomics of microsporeembryogenesis. Euphytica 2007a;158:275–85.

osp J, Tashpulatov A, Roessner U, Barsova E, Katholnigg H, Steinborn R, et al.Transcriptional and metabolic profiles of stress-induced, embryogenic tobaccomicrospores. Plant Mol Biol 2007b;63:137–49.

arillo JA, Pineiro M, Cubas P, Martínez-Zapater JM. Chromatin remodeling in plantdevelopment. Int J Dev Biol 2009;53:1581–96.

oosen R, Cordewener J, Supena EDJ, Vorst O, Lammers M, Maliepaard C, et al.Combined transcriptome and proteome analysis identifies pathways and mark-ers associated with the establishment of rapeseed microspore-derived embryodevelopment. Plant Physiol 2007;144:155–72.

ordan EG, Timmis JV, Trewaras AJ. The plant nucleus. In: Tolbert NE, editor. TheBiochemistry of Plants, vol. 1. New York, London: Academic Press; 1980. p.489–588.

afarga M, Casafont I, Bengoechea R, Tapia O, Berciano MT. Cajal’s contribution tothe knowledge of the neuronal cell nucleus. Chromosoma 2009;118:437–43.

afarga M, Berciano MT, García-Segura LM, Andrés MA, Carmo-Fonseca M. Acuteosmotic/stress stimuli induce a transient decrease of transcriptional activity inthe neurosecretory neurons of supraoptic nuclei. J Neurocytol 1998;27:205–17.

amond AI, Spector DH. Nuclear speckles: a model for nuclear organelles. Nat RevMol Cell Biol 2003;4:605–12.

iu J-L, Murphy C, Buszczak M, Clatterbuck S, Goodman R, Gall JG. The Drosophilamelanogaster Cajal body. J Cell Biol 2006;172:875–84.

ührmann R. snRNP proteins. In: Birnstiel ML, editor. Structure and Function of Majorand Minor snRNPs. Berlin, Heilderberg: Springer-Verlag; 1988. p. 71–99.

alik MR, Wang F, Dirpaul JM, Zhou N, Polowick PL, Ferrie AMR, et al. Transcriptprofiling and identification of molecular markers for early microspore embryo-genesis in Brassica napus. Plant Physiol 2007;144:134–54.

alik MR, Wang F, Dirpaul J, Zhou N, Hammerlindl J, Keller W, et al. Isolation ofan embryogenic line from non-embryogenic Brassica napus cv. Westar throughmicrospore embryogenesis. J Exp Bot 2008;59:2857–73.

araschin SF, de Priester W, Spaink HP, Wang M. Androgenic switch: an exam-ple of plant embryogenesis from the male gametophyte perspective. J Exp Bot2005;56:1711–26.

atera AG. Cajal bodies. Curr Biol 2003;13:R503–1503.elcak I, Cermanova S, Jirsova K, Koberna K, Malinsky J, Raska I. Nuclear pre-

mRNA compartmentalization: trafficking of released transcripts to splicingfactor reservoirs. Mol Biol Cell 2000;11:497–510.

onneron A, Bernhard W. Fine structural organization of the interphase nucleus insome mammalian cells. J Ultrastruct Res 1969;27:288.

oreno Díaz de la Espina S, Risueno MC, Medina FJ. Ultrastructural, cytochemicaland autoradiographic characterization of coiled bodies in the plant cell nucleus.Biol Cell 1982;44:229–38.

avascués J, Berciano MT, Tucker KE, Lafarga M, Matera AG. Targeting SMNto Cajal bodies and nuclear gems during neuritogenesis. Chromosoma2004;112:398–409.

latani M, Goldberg I, Swedlow JR, Lamond AI. In vivo analysis of Cajal body move-ment, separation, and joining in live human cells. J Cell Biol 2000;151:1561–74.

uvion-Dutilleul F, Besse S, Chan EK, Tan EM, Puvion E. p80-coilin: a compo-nent of coiled bodies and interchromatin granule-associated zones. J Cell Sci1995;108:1143–53.

uvion E, Lange M. Functional significance of perichromatin granule accumula-tion induced by cadmium chloride in isolated rat liver cells. Exp Cell Res1980;128:47–58.

uvion E, Viron A, Assens C, Leduc EH, Jeanteur P. Immunocytochemical identi-fication of nuclear structures containing snRNPs in isolated rat liver cells. JUltrastruct Res 1984;87:180–9.

t Physiology 168 (2011) 746–757 757

Raska I. Nuclear ultrastructures associated with the RNA synthesis and processing.J Cell Biochem 1995;59:11–26.

Raska I, Rychter Z, Smetana K. Fibrillar centers and condensed nucleolar chromatinin resting and stimulated human lymphocytes. Zeitschrift Fur Mikroskopisch-Anatomische Forschung 1983;97:15–32.

Raska I, Koberna K, Malinsky J, Fidlerova H, Masata M. The nucleolus and transcrip-tion of ribosomal genes. Biol Cell 2004;96:579–94.

Raska I, Andrade LE, Ochs RL, Chan EK, Chang CM, Roos G, et al. Immunological andultrastructural studies of the nuclear coiled body with autoimmune antibodies.Exp Cell Res 1991;195:27–37.

Risueno MC, Testillano PS. Cytochemistry and inmunocytochemistry of nucleolarchromatin in plants. MICRON 1994;25:331–60.

Schul W, Groenhout B, Koberna K, Takagaki Y, Jenny A, Manders EM, et al. The RNA3’ cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nucleardomains closely associated with coiled bodies and newly synthesized RNA.EMBO J 1996;15:2883–92.

Seguí-Simarro JM, Nuez F. How microspores transform into haploid embryos:changes associated with embryogenesis induction and microspore-derivedembryogenesis. Physiol Plant 2008;134:1–12.

Seguí-Simarro JM, Testillano PS, Risueno MC. Hsp70 and Hsp90 change their expres-sion and subcellular localization after microspore embryogenesis induction inBrassica napus L. cv Topas. J Struct Biol 2003;142:379–91.

Seguí-Simarro JM, Testillano PS, Risueno MC. MAP kinases are developmentally reg-ulated during stress-induced microspore embryogenesis in Brassica napus L.Histochem Cell Biol 2005;123:541–51.

Seguí-Simarro JM, Bárány I, Suárez R, Fadón B, Testillano PS, Risueno MC. Nuclearbodies domain changes with microspore reprogramming to embryogenesis. EurJ Histochem 2006;50:35–44.

Shaw PJ, Brown JWS. Plant nuclear bodies. Curr Opin Plant Biol 2004;7:614–20.Spector DL. Nuclear organization of pre-mRNA processing. Curr Opin Cell Biol

1993a;5:442–8.Spector DL. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol

1993b;9:265–315.Spector DL, Schrier WH, Busch H. Immunoelectron microscopic localization of

snRNPs. Biol Cell 1983;49:1–10.Spector DL, Fu XD, Maniatis T. Associations between distinct pre-mRNA splicing

components and the cell nucleus. EMBO J 1991;10-11:3467–81.Supena EDJ, Winarto B, Riksen T, Dubas E, van Lammeren A, Offringa R, et al.

Regeneration of zygotic-like microspore-derived embryos suggests an impor-tant role for the suspensor in early embryo patterning. J Exp Bot 2008;59:803–14.

Testillano PS, Coronado MJ, Seguí-Simarro JM, Domenech J, Gonzalez-Melendi P,Raska I, et al. Defined nuclear changes accompany the reprogramming of themicrospore to embryogenesis. J Struct Biol 2000;129:223–32.

Testillano PS, González-Melendi P, Ahmadian P, Risueno MC. The methylation–acetylation (MA) method, an ultrastructural cytochemistry for nucleic acidscompatible with immunogold studies. J Struct Biol 1995;114:123–39.

Testillano PS, González-Melendi P, Coronado MJ, Seguí-Simarro JM, Moreno MA,Risueno MC. Differentiating plant cells switched to proliferation remodelthe functional organization of nuclear domains. Cytogenet Genome Res2005;109:166–74.

Testillano PS, Sánchez-Pina MA, Olmedilla A, Fuchs JP, Risueno MC. Characteriza-tion of the interchromatin region as the nuclear domain containing snRNPs inplant cells. A cytochemical and immunoelectron microscopy study. Eur J CellBiol 1993;61:349–61.

Testillano PS, Sanchez-Pina MA, Olmedilla A, Ollacarizqueta MA, Tandler CJ, RisuenoMC. A specific ultrastructural method to reveal DNA: the NAMA-Ur. J HistochemCytochem 1991;39:1427–38.

Thiry M. The interchromatin granules. Histol Histopathol 1995;10:1035–45.Tykarska T. Rape Embryogenesis. III: Embryo development in time. Acta Societatis

Botanicorum Poloniae 1980;49:369–85.Vazquez-Nin GH, Echeverria OM, Ortiz R, Ubaldo E, Fakan S. Effects of hypophy-

seal hormones on transcription and RNA export to the cytoplasm. Exp Cell Res1997;236:519–26.

Visa N, Puvion-Dutilleul F, Bachellerie JP, Puvion E. Intranuclear distribution of U1

and U2 snRNAs visualized by high resolution in situ hybridization: revelation ofa novel compartment containing U1 but not U2 snRNA in HeLa cells. Eur J CellBiol 1993;60:308–21.

Williams M. Stereological techniques. In: Glauert AM, editor. Practical Meth-ods in Electron Microscopy. Amsterdam: North Holland/American Elsevier;1977.