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The Plant Cell, Vol. 15, 2253–2264, October 2003, www.plantcell.org © 2003 American Society of Plant Biologists

The Transport of Prolamine RNAs to Prolamine Protein Bodies in

Living Rice Endosperm Cells

Shigeki Hamada,

a,1

Keiki Ishiyama,

a,1,2

Sang-Bong Choi,

a,3

Changlin Wang,

a

Salvinder Singh,

a,4

Naoko Kawai,

a

Vincent R. Franceschi,

b

and Thomas W. Okita

a,5

a

Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164

b

School of Biological Sciences, Washington State University, Pullman, Washington 99164

RNAs that code for the major rice storage proteins are localized to specific subdomains of the cortical endoplasmic reticu-lum (ER) in developing endosperm. Prolamine RNAs are localized to the ER and delimit the prolamine intracisternal inclu-sion granules (PB-ER), whereas glutelin RNAs are targeted to the cisternal ER. To study the transport of prolamine RNAs tothe surface of the prolamine protein bodies in living endosperm cells, we adapted a two-gene system consisting of greenfluorescent protein (GFP) fused to the viral RNA binding protein MS2 and a hybrid prolamine RNA containing tandem MS2RNA binding sites. Using laser scanning confocal microscopy, we show that the GFP-labeled prolamine RNAs are trans-

ported as particles that move at an average speed of 0.3 to 0.4

�

m/s. These prolamine RNA transport particles generallymove unidirectionally in a stop-and-go manner, although nonlinear bidirectional, restricted, and nearly random movementpatterns also were observed. Transport is dependent on intact microfilaments, because particle movement is inhibited rap-idly by the actin filament–disrupting drugs cytochalasin D and latrunculin B. Direct evidence was obtained that these prola-mine RNA-containing particles are transported to the prolamine protein bodies. The significance of these results with re-gard to protein synthesis in plants is discussed.

INTRODUCTION

mRNA localization is a key mechanism in controlling the syn-thesis of proteins to specific regions of the cell. This process isinvolved in cell fate determination in yeast (Chartrand et al., 2001)and during early vertebrate development (Bashirullah et al., 1998;Palacios and Johnston, 2001). In somatic cells, RNA localizationmediates the targeting of proteins to specific subcellular regions,especially in structurally polarized cells (Ainger et al., 1997;Carson et al., 1998; Shestakova et al., 2001). RNA localizationalso is observed in plants during embryogenesis and polarized cellgrowth (Okita and Choi, 2002). This process likely is involved inthe intercellular transport of RNAs through the plasmodesmata(Haywood et al., 2002).

Rice synthesizes major quantities of two storage proteins,prolamines and glutelins. These proteins are synthesized on theendoplasmic reticulum (ER), translocated to the ER lumen, andthen packaged in separate compartments of the endomem-brane system. Prolamines are sequestered within the ER lumen

as intracisternal granules, whereas glutelins are transported viathe Golgi to storage vacuoles. We demonstrated previously thatthe rice storage protein RNAs are localized to distinct subdo-mains of the ER (Li et al., 1993; Choi et al., 2000; Hamada et al.,2003). Glutelin RNAs, which code for the major storage protein,are localized to the cisternal ER, whereas prolamine RNAs aretargeted to ER that surrounds the intracisternal prolamine pro-tein bodies (PB-ER). Prolamine RNAs are localized to the PB-ERby an RNA-targeting mechanism (Choi et al., 2000). Interestingly,although the prolamine primary sequence is not essential for RNAtargeting to the PB-ER, a translation AUG initiation codon isrequired. In the absence of an intact AUG, the prolamine RNAis mislocalized to the cisternal ER. These observations suggestthat the regulated prolamine RNA pathway requires the partici-pation of the translational machinery (Choi et al., 2000).

Results obtained from deletion studies of prolamine RNA indi-cate that PB-ER targeting requires the presence of two

cis

ele-ments (Hamada et al., 2003) or “zip codes” (Singer, 1993). Thepresence of a single zip code results in localization not only tothe PB-ER but also to the cisternal ER. These observations in-dicate that prolamine RNAs are directed to the PB-ER and thatthe absence of one or more zip codes results in RNAs trans-ported along a default pathway to the cisternal ER. Interestingly,despite the existence of this constitutive RNA transport to thecisternal ER, glutelin RNAs are localized to the cisternal ER by aregulated pathway whose RNA signal determinant apparently isdominant over the zip codes that direct prolamine RNA to thePB-ER (Choi et al., 2000; Hamada et al., 2003).

The storage protein RNAs are transported to a region of theendosperm cell that is rich in ER. Optical sectioning of the de-

1

These authors contributed equally to this work.

2

Current address: RIKEN Plant Science Center, 1-7-22 Suehiro-cho,Tsurumi-ku, Yokohama City, Kanagawa, 230-0045, Japan.

3

Current address: Department of Biological Sciences, Myongji Univer-sity, Yongin Kyunggido 449-728, Korea.

4

Current address: Department of Agricultural Biotechnology, AssamAgricultural University, Jorhat-785013, Assam, India.

5

To whom correspondence should be addressed. E-mail okita@wsu.edu;fax 509-335-7643.

Online version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.013466.

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2254 The Plant Cell

veloping endosperm cell by laser scanning confocal microscopyshows that the prolamine PBs are not distributed randomly inthe cytoplasm but are concentrated predominantly in the pe-ripheral regions of the cell (Muench et al., 2000). By contrast, theglutelin protein bodies are distributed throughout the cell, al-though in young cells they tend to cluster around the nucleus.The peripheral location of the prolamine PBs and their closeproximity to microtubules and microfilaments indicate that theseorganelles are associated with the cortical ER, a complex net-work of tubule and cisternal membranes (Muench et al., 2000).Hence, the majority of the storage protein RNAs are transportedfrom the nucleus, located centrally, or displaced to one end ofthe developing endosperm cell, to the peripheral cortex.

RNA transport is well characterized in several systems. In po-larized somatic cells and during early vertebrate development,endogenous RNAs are observed as large granules (Barbareseet al., 1995; Ainger et al., 1997; Carson et al., 1998; Rook et al.,2000; Krichevsky and Kosik, 2001) or particles (Sundell andSinger, 1990; Ferrandon et al., 1994; Forristall et al., 1995; Klocand Etkin, 1995) that move at rates ranging from 4 to 6

�

m/minvia cytoskeleton-associated motors (Bassell and Singer, 1997;Chartrand et al., 2001; Saxton, 2001; Kloc et al., 2002; Tekotteand Davis, 2002). When microinjected in oligodendrocytes, flu-orescently labeled myelin basic protein RNA first condenses intogranules that then move along microtubule tracks to the periph-eral extensions, where they are localized (Ainger et al., 1993). Mi-croinjection of the 3

�

untranslated region of bicoid RNA into the

Drosophila

embryo results in the recruitment of the RNA bindingprotein Staufen into large particles that are transported to theanterior pole by a microtubule-dependent process (Ferrandonet al., 1994, 1997). Thus, RNA transport occurs by a multistepprocess that involves granule formation, cytoskeleton-mediatedtransport, and localization (Wilhelm and Vale, 1993).

The nature of the RNA transport particle (granules) is begin-ning to emerge. In neurons, direct microscopic evidence has beenobtained showing that the RNA granules are large, tightly clus-tered aggregates of ribosomes. Hence, large translational com-plexes containing different RNA species are transported (Knowleset al., 1996; Krichevsky and Kosik, 2001; Mouland et al., 2001). Ithas been suggested that the formation of large RNA transport par-ticles occurs by the assembly of hundreds of copies of localizedRNAs and their corresponding RNA binding

trans

factors (Jansen,2001).

Recent developments in microscopy and the introduction offluorescent proteins coupled with recent advances in RNA bio-chemistry have resulted in the development of technology to mon-itor RNA movement and localization in living cells in real time(Bertrand et al., 1998; Takizawa and Vale, 2000). This technologytakes advantage of the green fluorescent protein (GFP) and thehigh affinity of specific RNA binding proteins (RBPs; e.g., MS2 coatprotein and U1A) to small 18- and 19-nucleotide stem-loop struc-tures. The RNA imaging system has two components: a GFP-RBPfusion protein and a hybrid RNA containing multiple copies of thestem-loop RNA binding sequences. When coexpressed, the GFP-RBP associates with the hybrid RNA, enabling one to monitorRNA transport by fluorescence microscopy in real time.

We have adapted the GFP monitoring system to follow thelocalization of rice storage protein RNAs in developing rice en-

dosperm as well as in heterologous tobacco BY-2 cells. Ex-pression in BY-2 cells established the utility of the two-genesystem for monitoring RNA transport. The MS2-GFP fusion pro-tein containing a nuclear localization signal (NLS) is found almostexclusively in the nucleus. When coexpressed with MS6X-prola-mine RNA, MS2-GFP is found mainly in the cell’s periphery, in-dicating nuclear export and transport to the cortical region, themain site of protein synthesis in plants. In developing endo-sperm expressing the two-gene system, prolamine RNAs arelocalized in particles that move to the surface of the prolaminePBs located in the cortical ER.

RESULTS

Localization of Prolamine mRNA Movement in TobaccoBY-2 Cells

To visualize native RNA transport during rice endosperm devel-opment, we adapted the GFP two-gene system developed byBertrand et al. (1998). In this approach, two synthetic genes areconstructed and coexpressed. One gene encodes a translationalfusion in which GFP is fused to the single-stranded RNA phagecoat protein MS2, which binds a defined 19-nucleotide RNAstem loop (Figure 1). The MS2-GFP protein also contains a NLSto restrict the protein to the nucleus if it is not complexed toRNA. The second gene encodes a hybrid RNA containing prola-mine RNA sequences fused downstream of the

�

-glucuronidase(GUS) coding sequence and tandem MS6X RNA binding sites.When the two genes are coexpressed, MS2-GFP binds to one ormore of the MS6X binding sites, which enables one to follow themovement of the RNA in real time by fluorescence microscopy.

The utility of this two-gene system in monitoring RNA trans-port was first assessed in tobacco BY-2 cultured cells. This wasaccomplished by inserting the 35S promoter of

Cauliflowermosaic virus

(CaMV) upstream of the NLS-MS2-GFP and GUS-MS6X-prolamine sequences and transforming these genes intoBY-2 suspension cells. Figure 2 shows the distribution of theGFP fusion protein, NLS-MS2-GFP, in BY-2 cells in the absence(Figures 2A to 2C) or the presence (Figures 2D to 2F) of the ex-pressed GUS-MS2-prolamine RNA. When expressed alone, theNLS-MS2-GFP gene is localized predominantly to the nucleus(Figure 2B). By contrast, the distribution of NLS-MS2-GFP whencoexpressed with the prolamine-MS6X RNA fusion is observedmainly at the cell’s periphery (i.e., in the cortical region) (Figure2E). Therefore, expression of the two-gene system resulted inthe nuclear export and transport of GFP-labeled RNA. Theseobservations readily support the feasibility of this approach inmonitoring RNA transport in plant cells.

Prolamine RNAs Form Particles in DevelopingRice Endosperm

To visualize prolamine RNA movement in developing rice en-dosperm cells, the promoters from the rice glutelin Gt1 (Zhenget al., 1993) and prolamine pProl (Wu et al., 1998) were insertedupstream of NLS-MS2-GFP and GUS-MS6X-prolamine, respec-tively (Figure 1). These two genes then were cloned into the T-DNAvector pCAMBIA1301, which was used to transform rice via

Agro-

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Prolamine RNAs in Rice Endosperm Cells 2255

bacterium tumefaciens

. Candidate plants for RNA movement werescreened initially by immunoblot analysis for the expression of thetwo-gene system (i.e., for the NLS-MS2-GFP fusion and the GUSprotein as well as for GUS histochemical staining activity inendosperm tissue). Plants that accumulated readily detectableamounts of these reporter proteins were selected and grownfor several generations before analysis. The results reportedhere were obtained from homozygous third- to fifth-generationplants.

Figure 3 show the distribution of the NLS-MS2-GFP fusionprotein in transgenic rice lacking GUS-MS6X-prolamine RNAsequences (Figures 3A to 3C) or coexpressing this prolaminetranscriptional gene (Figure 3D). When expressed alone, theNLS-MS2-GFP is distributed mainly in the nucleus in the en-dosperm sections (Figures 3A to 3C). When coexpressed withGUS-MS6X-prolamine RNA, an entirely different distributionpattern of fluorescence is seen. Numerous small fluorescentparticles ranging in size from 0.3 to 2

�

m in diameter are readilyevident (Figure 3D). These particles are distributed randomly inthe focal plane across the cell. Several fluorescent particlesmove within the observed focal plane of the cell, indicating

RNA movement. The movement patterns of this macromolecu-lar complex were investigated further as described below.

Movement of Prolamine RNA Transport Particles

Sections (75 to 100

�

m) of developing endosperm were placedon a glass slide, bathed in MS medium (Murashige and Skoog,1962) containing amino acids and sucrose, and observed by la-ser scanning confocal microscopy for RNA particle movement.Based on the observation of more than several hundred indi-vidual RNA transport particle movement patterns, the followingobservations are typical. Although many of the fluorescent par-ticles are stationary, other particles could be seen moving in andout of the focal plane of observation. In many instances, wewere able to capture the movement of particles within a focalplane, enabling its transport to be followed over time.

Figure 4 shows a series of snapshots taken at

�

15-s inter-vals depicting the movement of one particle. In time frame 1–3(0 to 45 s), the particle appears in the focal plane, where it re-mains stationary until between frames 4–6 and 7–8 (90 to 105s), where it commences nearly unidirectional movement. The

Figure 1. Scheme of the Two-Gene System to Visualize RNA Transport.

(A) The two-gene system consists of one gene that encodes a translational fusion between the viral MS2 coat protein (MS2) and GFP. The MS2-GFPtranslational fusion also contains an in-frame NLS from SV40, which targets the fusion protein to the nucleus. The second gene codes for a hybridGUS RNA that contains a 3� untranslated region consisting of an MS2 RNA binding site tandemly repeated six times and prolamine RNA. The GUSenzyme activity produced by this hybrid RNA together with GFP was used to select for transgenic plants.(B) When coexpressed, the NLS-MS2-GFP fusion protein interacts with the GUS-MS6X-prolamine RNA, enabling RNA transport to be monitored us-ing GFP fluorescence. To monitor RNA transport in developing rice endosperm cells, the prolamine Prol and glutelin Gt1 promoters were used to drivethe transcriptional and translational gene fusions, respectively. The CaMV 35S promoter was used to drive the expression of the two-gene system intobacco BY-2 cells.MS2, coat protein of bacteriophage MS; MS6X, hexa repeats of the MS2 RNA binding site; Pgt1, glutelin Gt1 promoter; Pprol, prolamine promoter;P35S, CaMV 35S promoter; 2x35S, CaMV 35S double enhancer; T35S, CaMV 35S terminator; Tagp, terminator of the rice ADP-glucose pyrophos-phorylase; Tnos, terminator of the nopaline synthase gene.

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2256 The Plant Cell

velocity of the particle, which assumes an elongated shape, isnot constant, for the particle moves in a saltatory manner. It re-mains stationary for several time frames before initiating move-ment again, eventually covering a distance of

�

30

�

m.A large fluorescent particle (labeled with an asterisk in frame

9–11) appears stationary in the first four frames. In subsequentframes, this large fluorescent particle apparently moves out ofthe focal plane (between frames 12–13 and 14–16), whereupon

the elongated particle transverses right over or very close to thesite occupied by the larger particle. In later time frames, theelongated particle moves very close to and nearly contacts asecond fluorescent particle, which remains stationary duringthis time sequence (see frames 24–25 and 26–28). The particlegoes on to move another 6

�

m before the fluorescent signalweakens and later disappears. The loss of the fluorescent sig-nal may be attributable to movement of the particle out of the

Figure 2. Assessment of Prolamine RNA Transport in Tobacco BY-2 Cells Using the Two-Gene System and Laser Scanning Confocal Microscopy.

(A) to (C) When expressed alone, the NLS-MS2-GFP fusion protein is located predominantly within the nuclei of BY-2 cells.(D) to (F) When coexpressed with GUS-MS6X-prolamine RNA, the fusion protein is distributed mainly to the cell’s periphery, indicating RNA-mediatednuclear export and transport to the cell’s cortical region.(A) and (D) Transmitted light images. Bars � 50 �m.(B) and (E) GFP fluorescence images.(C) Merged image of (A) and (B).(F) Merged image of (D) and (E).

Figure 3. GFP Localization in Developing Rice Endosperm Using the Two-Gene System as Viewed by Laser Scanning Confocal Microscopy.

(A) to (C) GFP fusion proteins are observed as large fluorescent clusters located in the nucleus when the NLS-MS2-GFP structural gene is expressedin the absence of the second GUS-MS6X-prolamine RNA coding gene. Nuclei were visualized by staining the endosperm section with propidium io-dide (A), and native GFP fluorescence is depicted in (B). (C) is a merged image of (A) and (B) showing the localization of the GFP fusion protein to thenucleus. Bar in (A) � 10 �m.(D) Expression of the two-gene system in developing rice endosperm results in the GFP fusion protein appearing as small fluorescent particles distrib-uted randomly within the microscopic focal plane.

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Prolamine RNAs in Rice Endosperm Cells 2257

focal plane. In several instances, however, the fluorescent sig-nal appears to expand with accompanying loss of fluorescence,a pattern suggesting that the particle dissociates.

Figure 5 shows the tracings of movement behavior of threeparticles that exemplify the velocity and directional movementpatterns typically observed in our study. Movement of the parti-cles generally is unidirectional, although changes in direction forshort distances also are observed, as shown in Figure 5A. Asecond typical characteristic of the movement of many particlesis that during transport, movement of the particle is restricted toa small area before commencing longer distance movement, asindicated in Figure 5B. Another example of these movement be-haviors can be seen in the supplemental data online (http://www.plantcell.org). A third type of particle movement is shown in Fig-ure 5C. The particle changes direction repeatedly, so that theoverall movement pattern appears stochastic.

The average velocities of these particles were estimated bymeasuring the distance traveled by the particle between twoadjacent frames, which ranged from a high of

�

1

�

m/s to a lowof less than

�

0.05

�

m/s. Despite the different patterns of move-ment shown in Figures 5A to 5C, the average velocities were sim-

ilar and varied only slightly between 0.3 and 0.4

�

m/s. This speedis consistent with that expected for the involvement of a cyto-skeleton-associated motor protein.

Prolamine RNA Particle Movement Is Dependent onIntact Microfilaments

To determine the role of the cytoskeleton, the movement behaviorof particles was investigated after treatment with different cyto-skeleton-disrupting drugs. Treatment of developing endospermsections for 20 min with 100

�

M nocodazole or 20

�

M latrunculinB disrupted the arrays of microtubules (Figures 6A and 6B) andactin filaments (Figures 6C and 6D), respectively, present in devel-oping endosperm. Actin filaments also were sensitive to cytocha-lasin D (data not shown). Developing endosperm sections show-ing particle movement were bathed in MS medium containing 100

�

M nocodazole, 20

�

M latrunculin B, or 100

�

M cytochalasin Dand examined for particle movement by laser scanning confocalmicroscopy. The earliest measurement for assessing RNA move-ment was at

�

3 to 5 min under the experimental conditions. Theresults of three different experiments are shown in Figure 7.

Figure 4. Movement of Prolamine RNA in Developing Rice Endosperm as Viewed by GFP Fluorescence Using the Two-Gene System and LaserScanning Confocal Microscopy.

Shown are snapshots taken at 15-s intervals depicting the movement of a prolamine RNA transport particle (arrows). The numbers represent the num-ber of 15-s time frames taken since the acquisition of the first image. Unlike most particles observed, this movement particle assumes an elongatedshape. The velocity of the particle is not constant; rather, it moves in a stop-and-go manner. In frames 9–11 and 12–13 (135 to 195 s), the particlemoves very near a large stationary particle (asterisk), which apparently moves out of the focal plane between frames 9–11 and 14–16 (165 to 210 s).Overall, the particles move a linear distance of �30 �m in 10 min before the fluorescence signal disappears. Bar � 10 �m.

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2258 The Plant Cell

Under normal conditions, particle movement could be de-tected easily for up to 30 min. The addition of DMSO, the sol-vent used to dissolve the cytoskeleton-disrupting agents, orthe microtubule drug nocodazole had minimal effect on RNAmovement except for one experiment in which particle move-ment was not observed after 9 min of treatment. By contrast, themicrofilament inhibitors cytochalasin D and latrunculin B rap-idly inhibited particle movement. Even at the earliest observa-tion time (3 to 5 min), particle movement was affected signifi-cantly by these microfilament inhibitors, because most particlesstopped moving and others simply oscillated very slowly in po-sition. In all cases, particle movement was inhibited completelywithin 5 min. These results are consistent with the view thatRNA particle movement is dependent on intact microfilaments.

Prolamine RNA Particle Transport to the ProlamineProtein Bodies

Prolamine RNAs are distributed specifically to the ER that boundthe prolamine PBs (Li et al., 1993; Choi et al., 2000). To identifythe location of the GUS-MS6X-prolamine RNA, in situ reversetranscriptase–PCR was performed using primers specific forGUS and Oregon Green dUTP followed by laser scanning con-focal microscopy to assess the distribution of the fluorescentlylabeled RNA (Figure 8). As expected, the GUS-MS6X-prolamineRNA (Figure 8A) is localized specifically to the prolamine PBs(Figure 8C), which are labeled preferentially with rhodamine hexylester (Figure 8B) as a result of its preferential binding to thehydrophobic prolamine polypeptides. These results indicate thatthe hybrid GUS-MS6X-prolamine RNAs are transported and lo-calized to the PB-ER.

Further evidence that GUS-MS6X-prolamine RNAs were trans-ported to the prolamine PBs was discovered by assessing thedistribution of the GFP-associated RNA transport particles in en-dosperm thick sections relative to prolamine PBs by observingnative GFP fluorescence in live endosperm sections. Becauserhodamine fluorescence (red emission) partially overlaps the greenportion of the spectra, the endosperm section was treated onlylightly with this vital stain by reducing the concentration and expo-sure time. Under these conditions, the amount of spillover byrhodamine hexyl ester fluorescence in the green channel wasnegligible compared with the native fluorescent signal emittedby GFP. Figure 8 shows the distribution of the RNA transportparticles as viewed by native GFP fluorescence (Figure 8D) rel-ative to prolamine PBs (Figure 8E) in a native endosperm sec-tion. Three GFP-containing RNA transport particles are evidentthat colocalize with prolamine PBs (Figure 8F). Not all of the pro-lamine PBs had associated RNA transport particles, indicatingthat the green fluorescent signal is not the result of spillover fluo-rescence from rhodamine hexyl ester staining.

Figure 5.

Variation in Particle Movement Patterns in Developing RiceEndosperm Cells.

The movement patterns of three particles were traced, and each timepoint was taken at 5-s intervals. These three types of movement weretypical of the hundreds observed during the course of this study andrepresent nearly unidirectional movement

(A)

, movement confined to a

small area

(B)

, and nearly random movement (constant changes in di-rection)

(C)

. Below each particle movement tracing is a graph depictingthe average velocity between two successive time points. Particle ve-locity ranges from a high of

�

1

�

m/s to a low of

�

0.05

�

m/s, with anaverage speed of 0.3 to 0.4

�

m/s.

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Prolamine RNAs in Rice Endosperm Cells 2259

Efforts were made to capture the movement of the RNA trans-port particle to the prolamine PBs in real time. Such a visual cap-ture of the transport of RNA particles to prolamine PBs was arare event. Figure 9 shows a tracing of the movement of an RNAtransport particle as viewed by GFP fluorescence. The particlemoves to a prolamine PB before the fluorescent signal fadesand disappears.

DISCUSSION

The two-gene system used in this study shows that RNA trans-port in plants shares many of the properties seen in other eu-karyotic systems (Wilhelm and Vale, 1993; Barbarese et al., 1995;Ferrandon et al., 1997; Bertrand et al., 1998; Wilkie and Davis,2001). First, RNA transport occurs via a large multi-macromolec-ular complex, a “particle.” Moving particles typically had an ob-servable size of

�

0.5 to 1

�

m in diameter, although larger parti-cles also were evident in developing rice endosperm. The actualsize of these particles likely is smaller because of the diffuse na-ture of the fluorescent signal (Barbarese et al., 1995) and theuse of six MS2 binding sites to amplify the fluorescent signal viathe interactive GFP fusion protein. The larger particles, whichmay be formed by the fusion of smaller particles, as seen in bud-

ding yeast (Bertrand et al., 1998), were stationary and had vari-able sizes up to 2

�

m in diameter. Some transport particles alsowere seen as elongated entities, as shown in Figure 4. These elon-gated particles appear to move by a process similar to that of aSlinky toy, in which one end serves as a pivot allowing the otherend to move and then pivot, thereby reinitializing the movementcycle.

Figure 7. Inhibition of Particle Movement by Cytoskeleton-DisruptingDrugs.

Developing endosperm sections (�75 to 100 �m thick) were bathed inamino acid– and sucrose-supplemented MS medium containing DMSO,100 �M nocodazole, 100 �M cytochalasin D, or 20 �M latrunculin B. Sec-tions then were viewed by laser scanning confocal microscopy for particlemovement for up to 30 min. The earliest observation took place 3 to 5 minafter the addition of the cytoskeletal inhibitor. The duration of particle move-ment is indicated by two connected circles. Control sections incubated insupplemented MS medium alone showed particle movement up to 20, 26,and 30 min in three separate experiments. The actin filament drugs cytocha-lasin D and latrunculin B rapidly stopped particle movement within 3 to 5min, whereas the microtubule inhibitor nocodazole had little effect. No parti-cle movement was evident after 3 to 5 min in endosperm sections treatedwith cytochalasin D in experiments 2 and 3 or with latrunculin B in experi-ment 3. Even when particle movement was detected in the presence ofthese microfilament inhibitors, as denoted by single circles, many of the par-ticles stopped moving while the rest oscillated in position very slowly.

Figure 6. Structure of Microtubules and Microfilament Arrays in Devel-oping Rice Endosperm and Their Disruption by Nocodazole and Latrun-culin B.

(A) Microtubules were visualized by indirect immunofluorescence label-ing methods using �-tubulin monoclonal antibody followed by fluores-cein-tagged goat anti-mouse antibody.(B) and (D) Preincubation of developing endosperm sections (�300 �mthick) in 100 �M nocodazole (B) or 20 �M latrunculin B (D) for 20 minresults in the disruption of these cytoskeletal elements.(C) Microfilaments were visualized directly with phalloidin-conjugatedAlexa Fluor 488.Bars � 50 �m.

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2260 The Plant Cell

Particle formation also was evident in tobacco BY-2 cells, al-though such particles were much larger (

�

2 to 3

�

m in diame-ter) than those seen in developing rice endosperm. In severalinstances, two or three closely associated particles were ob-served to move back and forth in unison (data not shown). Al-though there are several explanations that could account forthe particle size differences between the two cell types, theyare likely the result of differences in expression of the two-genesystem (Bertrand et al., 1998). The potent modified CaMV 35Spromoter from pRTL2, which contains double enhancer elementsand a TEV leader, was used to drive GUS-MS6X-prolamine RNAexpression in BY-2 cells, whereas the rice seed–specific prola-mine Prol promoter was used to drive the expression of this genein developing rice endosperm cells. In our hands, the Prol pro-moter was found to be a relatively weak promoter (our unpub-lished observations), especially compared with the Gt1 promoter,which is one of the strongest rice seed–specific promoters avail-able (Zheng et al., 1993). Increased constitutive expression hasbeen suggested to account for the formation of a single large par-ticle containing

ASH1

RNA instead of the several smaller particlesevident under controlled expression (Bertrand et al., 1998).Hence, the smaller particles in rice endosperm cells likely are at-tributable to the lower transcript levels of GUS-MS6X-prolamineRNA compared with the levels expressed in BY-2 cells.

A common feature of RNA localization is their final destinationsite in the plant cell. In tobacco BY2 cells and developing riceendosperm, RNAs are localized to the cortical region. In bothcell types, RNA transport particles are not prevalent in the peri-nuclear region. Such a condition should be readily conspicuousin BY-2 cells because of their highly vacuolated nature. Otherthan their predominant location at the cell’s periphery, fluores-cent RNA particles are evident on the cytoplasmic transvacuolarstrands. Because these “strands” contain actin filaments, the as-sociation of RNA particles with transvacuolar strands may repre-sent their transport via actomyosin (see below).

The transport and localization of RNAs to the cortical regionmay be essential for the expression of these gene sequences in

plants. Other than the perinuclear region and cytoplasmic trans-vacuolar strands, the cortical region is the only intracellular siterich in actin filaments. These cytoskeletal elements have beensuggested to be essential for efficient protein synthesis becausethey may provide a scaffold for polysomes or serve as a site en-

Figure 8. Prolamine RNA Transport Particles Are Transported to the Prolamine PBs.

(A) The distribution of GUS-MS6X-prolamine RNAs was visualized by confocal microscopic analysis of endosperm sections subjected to in situ re-verse transcriptase–PCR in the presence of GUS primers and Oregon Green-488 dUTP.(B) The same section stained with rhodamine hexyl ester, which specifically labels the prolamine PBs as a result of the affinity of this dye for the hy-drophobic prolamine polypeptides.(C) A merged image of (A) and (B) depicting the localization of the GUS-MS6X-prolamine RNAs to prolamine PBs. Bar � 10 �m.(D) to (F) Colocalization of GFP-tagged particles (F) as viewed by native GFP fluorescence (D) with prolamine PBs stained with rhodamine hexyl ester(E). Note that only three of the five prolamine PBs seen in these images show colocalization with GFP-tagged particles, indicating that green fluores-cence is not the result of spillover from rhodamine hexyl ester staining. Bar � 10 �m.

Figure 9. Movement of a Prolamine RNA Transport Particle to a Prola-mine PB.

A developing endosperm section was stained with rhodamine hexyl es-ter, washed to remove excess stain, and then analyzed for native GFPfluorescence by laser scanning confocal microscopy. Images were cap-tured at 15-s intervals. The numbers represent the number of 15-s timeframes taken since the acquisition of the first image. The distance be-tween the start and the final destination is 21 �m. A video depicting thistransport is available in the supplemental data online (http://www.plant-cell.org). Bar � 10 �m.

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riched for translation factors, such as eEF1A (Lenk and Penman,1979; Davies et al., 1991, 1996; Hesketh and Pryme, 1991).

The visualization of RNA movement in real time provides sev-eral insights into the localization mechanism. The movement ofthe RNA particle generally was directional, although nonlinearbidirectional movement also was seen. In many instances, theparticles transverse a wandering path whose total length spansmuch longer than the shortest distance between two points. Ir-respective of the path taken, the transport particles move withan average speed of �0.3 to 0.4 �m/s, a value similar to the es-timated velocity of �-actin RNA in fibroblasts (Oleynikov andSinger, 2003). The highest instantaneous velocity measured was1 �m/s, slower than the peak velocities (2 to 10 �m/s) observedfor the movement of the Golgi apparatus (Boevink et al., 1998;Nebenfuhr et al., 1999) and peroxisomes (Jedd and Chua, 2002)in plant cells. At least part, if not all, of the apparent differencesin peak velocities between the RNA particles and plant or-ganelles is the result of our inability to capture images by con-focal microscopy at a sufficient rate to accurately measure peakvelocities. In this study, RNA movement images were capturedat one frame every 5 s, a time frame that includes periods of dis-continuous movement behavior. By contrast, the estimation ofplant organellar movement was obtained at rates of one frameper second or greater (Boevink et al., 1998; Nebenfuhr et al., 1999;Jedd and Chua, 2002).

The movement patterns and average speeds of the RNA trans-port particles are consistent with the involvement of a cytoskeletalmotor protein (Bassell et al., 1999; Jansen, 1999, 2001; Tekotteand Davis, 2002). Indeed, RNA particle movement is inhibited bylatrunculin B and cytochalasin D, two drugs that disrupt microfila-ments. A specific motor protein, Myo4, an unconventional, non-muscle class-V myosin, is essential for the transport of ASH1RNA to the daughter cell in budding yeast (Long et al., 1997;Takizawa et al., 1997). Interestingly, analysis of the Arabidopsisgenome identified 17 myosin sequences (Reddy and Day, 2001).Surprisingly, none of these myosins belongs to class V; instead,they fall into class VIII and class XI, myosin types unique to plantspecies. Myosin XI has many structural features similar to thoseof myosin V, including six light chain binding motifs (IQ motifs)and a C-terminal tail with coiled-coil segments interspersed withnonhelical regions (Reck-Peterson et al., 2000). Based on struc-tural similarities, it is likely that a member of this myosin classplays a role in RNA transport.

In addition to the yeast ASH1 and the rice storage proteinRNAs, the only known examples of RNA transport by actomyo-sin involve �-actin in fibroblasts and possibly prospero mRNAin Drosophila neuroblasts (Palacios and Johnston, 2001). Thebulk of the RNAs transported in animal cells are microtubulebased (Pokrywka, 1995; Bogucka-Glotzer and Ephrussi, 1996;Carson et al., 1997; Arn and Macdonald, 1998; Bloom and Beach,1999; Tekotte and Davis, 2002). The use of microtubules in RNAtransport may reflect the fact that this cytoskeleton component iscapable of forming long structures, especially in polarized, differ-entiated cell types of vertebrate cells and oocytes (Steebings,2001).

Ongoing studies in this laboratory indicate the existence ofmultiple RNA transport pathways from the nucleus to the corti-cal region in developing rice endosperm cells (Hamada et al.,

2003). In addition to the prolamine RNA pathway to the PB-ER,there exists a glutelin RNA pathway as well as a constitutivepathway to the cisternal ER. Analysis of glutelin RNA transportusing the two-gene system reveals no apparent differences inRNA movement from that seen for prolamine RNAs (our unpub-lished observations). Glutelin RNAs are transported as particleswith movements similar, if not identical, to those describedhere for prolamine RNA transport (data not shown). The forma-tion of a particle may be a requisite step for RNA transport tothe cortical region in developing rice endosperm. Although notstudied directly, it is likely that RNAs engaged in the constitu-tive pathway are transported and localized to the cortical re-gion by a process similar to that seen for the storage proteinRNAs. The formation of the particle is likely to involve general-ized localization elements that direct transport to the corticalER and specialized signal determinants (zip codes) that targetthe RNA to specific subdomains of the cortical ER. These spe-cialized cis elements or zip codes have been identified for theprolamine RNA and are sufficient to direct reporter RNAs to thePB-ER (Hamada et al., 2003). The glutelin 3� untranslated re-gion also has one or more specialized cis elements to direct theRNA to the cortical ER (Choi et al., 2000; Hamada et al., 2003).

The intensity of the fluorescent signal and the size of the par-ticle suggest that multiple RNAs are cotransported withinthe same entity (Fusco et al., 2003). In Drosophila, bicoid RNAforms a particle in conjunction with Staufen, a helicase-like pro-tein that contains five double-stranded RNA binding domains.The recruitment of Staufen into particles is dependent on inter-molecular RNA–RNA interactions, suggesting that the particleis a large multimolecular complex containing many RNAs(Ferrandon et al., 1997). Microinjected fluorescently labeled wing-less and pair-rule transcripts coassemble into a particle and moveto the apical end of the Drosophila blastoderm along microtu-bules (Wilkie and Davis, 2001). The presence of multiple RNA spe-cies within a transport particle also is supported by the cotrans-port of fluorescently tagged Human immunodeficiency virus (HIV)RNAs in oligodendrocytes. Both the HIV gag and vpr RNAs con-tain the A2RE zip code that is recognized by hnRNP A2, which me-diates anterograde transport of the A2RE-containing RNA granules(particles) along microtubules (Barbarese et al., 1995; Ainger et al.,1997; Carson et al., 1997). Based on ratiometric analysis, eachtransport particle was estimated to contain 29 molecules of thesefluorescently tagged RNA species (Mouland et al., 2001). This esti-mated RNA content probably is much lower than the actual levels,because the transport RNA granules likely contain other endoge-nous unlabeled A2RE-containing RNAs. Studies now under wayare aimed at isolating these GFP-tagged RNA transport parti-cles and assessing their RNA composition.

METHODS

Plasmid DNA Construction and Plant Transformation

To visualize the movement of prolamine RNAs in living cells, the two-gene system from Bertrand et al. (1998) was adapted. Plasmid express-ing the MS2-GFP translational fusion gene in BY-2 tobacco (Nicotianatabacum) cells was obtained as follows. GFP (Davis and Vierstra, 1998)

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DNA coding sequence was amplified using primers (5�-GTATCAGCG-GCCGCGAGTAAAGGAGAAGAACTT-3� and 5�-GAAATTCGAGCTCTT-ATTTGTATAGTTCATCC-3�) and then cloned into the NotI-SalI sites ofpBluescript KS II� (Stratagene). A BamHI-NotI fragment encoding anSV40 nuclear localization sequence and MS2 viral capsid protein wasobtained from pG14-MS2-GFP (Bertrand et al., 1998) and cloned intothe upstream BamHI-NotI site of the GFP sequence. The resulting NLS-MS2-GFP was removed with BamHI and SacI digestion and inserted intopBluescript KS II� containing the nos 3� terminator (Tnos) cloned intothe SacI and EcoRI sites. The NLS-MS2-GFP-Tnos DNA fragment thenwas obtained by digestion with BamHI and HindIII and transferred intopET30a() (Novagen, Madison, WI). The 35S promoter of Cauliflowermosaic virus (CaMV) was cloned into the NcoI and BamHI sites ofpET30a() containing NLS-MS2-GFP-Tnos. The CaMV 35S-NLS-MS2-GFP-Tnos DNA then was generated by digestion with BglII and HindIIIand subsequently cloned into the BamHI and HindIII sites of pCAMBIA1300to produce a control plasmid, pSB20.

To generate the hybrid RNA gene fusion consisting of �-glucuronidase(GUS)-MS6X-prolamine, prolamine7 cDNA was isolated by digestion ofpProl7 (Kim et al., 1988) with HincII and NotI and then cloned into theEcoRV and NotI sites of pSL-MS2-6 (Bertrand et al., 1998). The resultingplasmid then was digested with BamHI, and the MS6X-prolamine se-quences were cloned subsequently at the filled-in XhoI-NcoI sites down-stream of the GUS coding sequence of pRTL2 to produce CaMV 35S2X-GUS-MS6X-T35S. This transcriptional fusion gene was obtained byHindIII digestion, and the resulting fragment was cloned into the samesite of pSB20 to yield pSB21 for prolamine.

pSB15 and pSB16 were constructed for expression in rice (Oryza sa-tiva) endosperm cells. A NotI-SalI fragment containing the GFP genefrom pG14-MS2-GFP (Bertrand et al., 1998) and a ClaI-HpaI fragmentcontaining the ADP-glucose pyrophosphorylase terminator (Tagp) werecloned into the NotI-HindIII and ClaI-HincII sites, respectively of pBlue-script II KS. The BamHI-NotI insert from pG14-MS2-GFP containing theNLS-MS2 fusion was cloned into pSL1180 (Pharmacia). To combineNLS-MS2 and GFP-Tagp, a NotI-XhoI fragment containing GFP-Tagpwas cloned into the same sites of pSL1180 containing the NLS-MS2 se-quence. A BamHI-BglII fragment containing NLS-MS2-GFP-Tagp thenwas transferred into the BglII site of pSP72, which contains the 1.8-kbGt1 promoter fragment (Zheng et al., 1993) inserted at the EcoRI-BglIIsites (Choi et al., 2000).

The RNA transcriptional gene fusion was driven by the prolaminePprol promoter (Wu et al., 1998) and was prepared as follows. A SacI-HindIII fragment containing the nos terminator (Tnos) was cloned intopSL1180. To form the Pprol-MS6X fusion, the MS6X fragment (BamHI-SacI) from pSL-MS2-6 was cloned into the BamHI-SacI of pProl (Wu etal., 1998). The resulting plasmid was digested with EcoRI and NotI, andthe Pprol:MS6X fragment was cloned upstream of Tnos. The GUS se-quence was amplified using the primers 5�-GAGGATCCCCGGGTAGGT-CAGTCCC-3� and 5�-CAGGATCCTTGTTGATTCATTGTTTGC-3� andcloned into the BamHI site after digestion of Pprol:MS6X:Tnos. Prola-mine cDNA that had been digested with HincII-NotI was cloned into theHpaI-NotI sites of the plasmid containing Pprol-GUS-MS6X-Tnos toyield Pprol-GUS-MS6X-prolamine cDNA-Tnos.

The MS2-GFP and GUS-MS6X-prolamine-Tnos DNA fragments werecombined into a single plant expression vector, pCAMBIA1300. TheXbaI-BglII fragment containing the MS2:GFP gene was cloned into theXbaI and BamHI sites of pCAMBIA1300 and designated pSB15. AHindIII fragment containing Pprol-GUS-MS6X-prolamine cDNA-Tnoswas inserted into the HindIII site of pSB15 to produce pSB16.

Transformation of tobacco BY-2 calli was achieved by cocultivationwith Agrobacterium tumefaciens Agl1 for 3 days at 28C. Calli then weretransferred onto an MS medium plate (Murashige and Skoog, 1962) con-taining 0.2 mg/L 2,4-D, 150 �g/mL timentin, and 50 �g/mL hygromycin.Cell suspension lines were prepared by growing calli on liquid MS selec-

tion medium. Transformation of rice and rice growth conditions were asdescribed previously (Choi et al., 2000).

In Situ Localization of GUS-MS6X-Prolamine RNA

In situ reverse transcriptase–PCR was conducted with tissue sectionsfrom developing seeds of pSB16 plants using specific primers for GUS(sense, 5�-CAGCGAAGAGGCAGTCAACGGGGAA-3�; antisense, 5�-CATTGTTTGCCTCCCTGCTGCGGTT-3�) as described previously (Choiet al., 2000). The initial reverse transcriptase reaction cycle was per-formed for 3 min at 94C, 20 min at 60C, and 5 min at 72C, which wasfollowed by eight amplification cycles at 94C for 30 s, 58C for 1 min,and 72C for 90 s.

Tissue Preparation and Confocal Microscopy

Tobacco BY-2 suspension-cultured cells were mounted on microscopeslides in MS medium and observed using a standard fluorescein filterset. Confocal images were obtained on a Zeiss 410-series laser scan-ning confocal microscope (Jena, Germany) and a Bio-Rad View ScanVDC-250 laser scanning confocal microscope using a �40 objective.Untransformed BY2 cells were imaged to set the lower limits of detectionfor GFP fluorescence without interference from native autofluorescence.

Glumes from developing rice seeds were removed by hand. Thick sec-tions (75 to 100 �m) from 10- to 14-day-old rice seeds were obtained us-ing a vibratome and mounted on a microscope slide in MS medium con-taining an amino acid mixture and 2% sucrose using a recipe developedby Donovan and Lee (1977). Confocal analysis was performed using thesame conditions used for tobacco except that a �60 objective wasused. Developing sections from wild-type plants were used to set thelower limits of detection for GFP fluorescence without interference fromnative autofluorescence.

Cytoskeletal Inhibitor Studies

Sections of developing endosperm were obtained and bathed in MS me-dium containing amino acids and 2% sucrose (Donovan and Lee, 1977)and nocodazole, latrunculin B, or cytochalasin D. The cytoskeletal inhibi-tors were prepared as concentrated (100 to 200 times) stock solutions inDMSO and stored at �20C. The endosperm sections then were observedcontinuously for particle movement by laser scanning confocal micros-copy for up to 30 min. Sections treated with MS medium containing anequivalent concentration of DMSO (5%, v/v) were used as controls.

Immunofluorescence Studies for Microtubules and Actin Filaments

The glumes of 12- to 18-day-old developing rice seeds were removed byhand. The developing seeds were sectioned by free hand into 300-�m-thick specimens. The sections were placed immediately in a Teflon-coated depression slide (Electron Microscopy Sciences, Fort Washing-ton, PA) containing 150 �L of cytoskeleton-stabilizing buffer (CSB; 50mM Pipes, pH 6.9, 5 mM EGTA, 2 mM MgSO4, 1.0% DMSO, 0.1% TritonX-100, and 200 �M phenylmethylsulfonyl fluoride) containing variousamounts of nocodazole or latrunculin B. After 20 min of incubation atroom temperature, the CSB solution was replaced with 100 �M cross-linking agent (m-maleimidobenzoyl-N-hydroxysuccinimide ester [Pierce,Rockford, IL] in CSB) and incubated for another 20 min. The sectionsthen were fixed with freshly prepared 4% formaldehyde solution in CSBfor 1 h and washed three times in PBS. Sections were incubated with cellwall digestion solution (1% cellulose, 0.4 M mannitol, 0.1% Triton X-100,0.3 mM phenylmethylsulfonyl fluoride, and 5 mM EGTA, pH 5.5) at 37Cfor 3 min followed by a 15-min wash in PBS supplemented with 0.1%

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Triton X-100 (PBST). The sections then were blocked in PBS containing2% BSA and 0.1% Triton X-100 for 45 min at 37C.

To visualize microtubules, the permeabilized sections were incubatedwith mouse monoclonal anti-�-tubulin antibody (N356; Amersham) di-luted 300-fold in blocking buffer overnight at 4C. The sections werewashed three times with PBST and then incubated for 1 h with fluores-cein-conjugated goat anti-mouse antibody diluted 300-fold. To visualizeactin filaments, the permeabilized sections were incubated directly in330 nM Alexa 488 phalloidin in PBS for 1 h. The sections were washedwith PBST and twice with PBS. A drop of FluoroGuard Anti-Fade reagent(Bio-Rad) was added to the sections and examined by laser scanningconfocal microscopy as described (Muench et al., 2000).

Upon request, materials integral to the findings presented in this pub-lication will be made available in a timely manner to all investigators onsimilar terms for noncommercial research purposes. To obtain materials,please contact T. W. Okita, okita@wsu.edu.

ACKNOWLEDGMENTS

We thank Robert H. Singer (Albert Einstein College of Medicine, Bronx,NY) for the generous use of pG14-MS2-GFP and pSL-MS2-6, JamesCarrington (Oregon State University, Corvallis, OR) for pRTL2 plasmid,and Fumio Takaiwa (National Institute of Agrobiological Resources,Tsukuba, Japan) for the prolamine promoter Pprol. This work was sup-ported by grants from the National Science Foundation.

Received May 9, 2003; accepted July 11, 2003.

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DOI 10.1105/tpc.013466; originally published online September 24, 2003; 2003;15;2253-2264Plant Cell

Vincent R. Franceschi and Thomas W. OkitaShigeki Hamada, Keiki Ishiyama, Sang-Bong Choi, Changlin Wang, Salvinder Singh, Naoko Kawai,

The Transport of Prolamine RNAs to Prolamine Protein Bodies in Living Rice Endosperm Cells

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