Plant Physiol. (1992) 100, 1627-16320032-0889/92/100/1 627/06/$01 .00/0

Received for publication August 12, 1992Accepted September 21, 1992

Nuclear Targeting in Plants'

Natasha RaikhelDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312

The nucleus is the site of highly active two-way macro-molecular traffic between the nucleoplasm and the cyto-plasm. Nuclear import and export must be highly specificprocesses because the content of the nucleus is distinguishedcompared to the cytoplasm. This is rather amazing, consid-ering that the nuclear envelope dissolves during mitosis ofanimal and plant cells, necessitating reassembly of all nuclearcomponents and reentry of nuclear proteins into the nucleus.In addition, many basic cellular processes, such as genetranscription and cell division, clearly require proper nuclearlocalization of regulatory and housekeeping proteins. Fur-thermore, experimental evidence indicates the existence ofreceptors and other cellular factors that mediate nucleartransport. Nuclear transport, although extensively studied inanimal and lower eukaryotic systems (3, 20), has only recentlybeen addressed in higher plants (1, 2, 5, 7, 8, 15, 24-26).This attention to protein and nucleic acid transport into thenucleus in plants was stimulated not only by a need tounderstand basic cell biology, but also by the desire to mod-ulate or regulate the expression of genes via geneticengineering.The nucleus (Fig. 1) is surrounded by the nuclear envelope,

which includes the inner and outer nuclear membranes and,between them, the perinuclear space. The outer nuclear mem-brane is contiguous with the rough ER, and its outer surfaceis generally studded with ribosomes. Underlying the innernuclear membrane is the nuclear lamina, which is composedof a network of filamentous proteins. Unlike other organelles,the nuclear envelope contains NPC2, the major sites of bidi-rectional protein and nucleic acid traffic. Though understoodon a structural level, knowledge of the biochemistry behindthe NPC is limited. Only a few proteins associated with theNPC in animal systems have been identified (3); however,their actual involvement in nuclear import has not beendemonstrated.

Structurally, the NPC traverses the double membrane,bringing the lipid bilayers of the inner and outer membranestogether around the margins of each pore. The NPC consistsof eight large protein granules arranged in a circle with alarge central granule or transporter that is thought to controlactive nucleocytoplasmic transport of large molecules (6). In

1 Support was from the United States Department of Energy,Washington, DC.

2Abbreviations: NPC, nuclear pore complexes; NLS, nuclear lo-calization signal; SV40, simian virus 40; MAT a2, mating type a2;GUS, fl-glucuronidase; 02, Opaque-2; b-ZIP, basic/leucine zipper;NEM, N-ethylmaleimide; NBP, nuclear binding protein.

addition, eight peripheral channels of a smaller diameterwould allow a passive exchange of small molecules. There-fore, transport into the nucleus is fundamentally differentfrom that into other organelles, where transported proteinspass directly through the membrane. Although most nuclearproteins carry information that simply directs them throughthe nuclear pore, others must continue their journey to a finalsubnuclear compartment (12). With regard to what is specif-ically responsible for proper localization of a protein insidethe nucleus, very little is known.

Results obtained from yeast and animal systems have ledinvestigators to suggest some basic rules for nuclear proteintargeting (3). Proteins smaller than 40 to 60 kD are thoughtto diffuse through the nuclear pore, though no physiologi-cally relevant macromolecule has been shown to traverse thenuclear pore by diffusion. However, it has been shown thatmany proteins require at least one NLS. Two criteria are usedto define these NLSs: (a) an NLS is sufficient to redirect acytoplasmic protein to the nucleus, and (b) an NLS is neces-sary for directing a nuclear protein to the nucleus. However,in many cases the NLSs are not adequately characterized byboth criteria.Known NLSs can be grouped into several categories (3).

First, SV40-like NLSs contain a short stretch of basic aminoacids (PKKKRKV). Second, MAT a2-like NLSs consist ofshort hydrophobic regions that contain one or more basicamino acids (KIPIK). Third, bipartite NLSs are usually acombination of two regions of basic amino acids separatedby a spacer of more than four residues (SPPKAVKRPA-ATKKAGQAKKKKLDKEDES) (17). The first region has twobasic amino acids and the second has at least three out offive basic residues. According to current speculation, the twobasic regions cooperate in binding, whereas the spacer mayfacilitate their cooperative interaction. In addition, there aresome NLSs that do not fit any of the above-mentioned groupsand that are characteristic of some viral nuclear proteins (forexample, the NLS of influenza ribonucleoprotein is AA-FEDLRVRS). Although NLSs have been grouped into thesecategories, a consensus as to sequence, either between orwithin groups, has not been reached.

This article will focus on recent advances in our under-standing of NLSs recognized in plants. For earlier reviews onnuclear targeting in animal and yeast cells, see Garcia-Bustoset al. (3) and Silver (20).

THE SV40-TYPE NLS IS RECOGNIZED IN PLANTS

One question addressed recently is whether or not theSV40-type sequence can be recognized in plants. The SV40

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Plant Physiol. Vol. 100, 1992

RER

/1'.

nuclear porecomplexes

NPC:

centralgranule

nucleolusNPC

nuclearlamina

chromatinFigure 1. Schematic representation of the nucleus.

NUCLEAR LOCALIZATION SUFF.PROTEIN SIGNALS NEC. REF.

TGA-IA (tobacco)a LAQNREA A_ +/NA 24

TGA-1B (tobacco) ILVRNRESAQLS +/NA 24

02 NLS B (maize)r +/NA 26

Nla (Potvirus)' + /KHKLKM-32aa ++ 1

VIrD2 3PREDDDGEPS S +/+ 7, 22(A"robactenum)rVIrE2 NSE1 UPEDRYIQTEYG + /+ with NSE2 2(4grobaterwm)aVIrE2 NSE2 ITYGSDTEIPSg +/+ with NSE1 2(Agbacterium)a02 NLS A (maize)b MEEAVTMAPAAVSSAWGDP +/NA 26

MEYNAIUELEEDLER NLS A (maize)b GDWAAP -/+ with NLS M 19. NLS M (MaiZe)b MSlMIM +/+ with NLS A 19

or C

. NLS C (maize)b MISEALUAIGU +/+ with NLS M 19

Figure 2. Nuclear localization signals identified in plants. aPredictedbipartite NLSs from experimentally tested peptides are shown.bExperimentally tested peptides are shown. SUFF., Signal is suffi-cient for redirection of a reporter protein to the nucleus; NEC.,signal is necessary for nuclear protein import; NA, not analyzed.

sequence (PKKKRKV) was fused to a reporter protein, a 69-kD GUS, and the construct was transformed into tobacco. Byhistochemical analysis of GUS, it was shown that a seven-amino acid SV40 sequence can function as an NLS in trans-genic tobacco plants (24), but a mutant SV40 NLS(PKTKRKV), in which an essential K was mutated to T,cannot. Nuclear localization was also observed when the100-kD T7 RNA polymerase protein was fused to the SV40NLS and electroporated into tobacco protoplasts (8). Theseresults clearly indicate that some mechanisms of nucleartransport are common between animal and plant cells.

Indeed, some nuclear targeting sequences in plant DNA-binding proteins have been identified by their similarity tothe SV40 NLS (24). Three DNA-binding proteins, TGA-1A,TGA-1B, and TFIID were tested to see whether or not theycould facilitate nuclear import of a GUS reporter protein.Regions selected by clusters of basic amino acids from theseproteins were fused to an amino-terminal portion of GUS,and the cellular location of GUS was analyzed using histo-chemical staining in transgenic tobacco plants. Twenty-fouramino acids of TGA-1B and 70 amino acids of TGA-1A, bothlocated in the basic DNA-binding domain of these proteins,were able to function as nuclear targeting signals (Fig. 2).However, the 25-amino acid basic region of the DNA-bindingprotein TFIID fused to GUS shows staining only of thecytoplasm in transgenic tobacco plants. It was suggested thatthe TFIID protein, which has a molecular mass of 23 kD,

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TRANSPORT OF PROTEINS TO THE NUCLEUS

either passively moves into the nucleus or is directed thereby an NLS located in an unanalyzed part of the protein.

Both means of transport are possible: some small proteinshave been shown to be passively transported to the nucleusvia cotransport with another protein (20), but other smallproteins, such as the 13.8-kD heat shock protein in yeast (13)and the 28-kD high-mobility group 1 protein from calf thy-mus (23), have been shown to have an NLS capable ofredirecting a reporter protein. Also, there have been severalcases in which sequences very similar to the SV40 NLS failto function in nuclear targeting (20). Thus, the experimentswith SV40-type NLSs clearly demonstrate that this is a signalthat is recognized by the plant nuclear import machinery.However, not all sequences reminiscent of the SV40-type areutilized as NLSs.

NUCLEAR LOCALIZATION OF TWO REGULATORYPROTEINS FROM MONOCOTS

Tight control must be kept over nuclear protein localizationbecause regulatory proteins are required in the nucleus onlyat very specific developmental times and only in specifictissues. It is quite possible that regulatory proteins are keptin the cytoplasm until their function in the nucleus is needed.The availability of two monocot regulatory proteins, 02 andR, sparked our analysis of their nuclear import mechanism.

NLSs of the 02 Protein

The maize trans-acting factor, 02, regulates the expressionof 22-kD zein genes (18) and has been shown to localize tothe nucleus in maize endosperm tissue and in transformedtobacco plants, indicating that the protein nuclear importmachinery is similar in monocots and dicots (25). Althoughthe 47-kD 02 protein may be small enough to diffuse throughthe nuclear pores, it was demonstrated that fusion proteinconsisting of 02 fused to GUS is sufficient to direct GUSprotein to the nucleus in transgenic tobacco cells and intransiently transformed onion cells (26). Two independentregions of 02 were identified as being able to redirect GUSto the nucleus in both systems. A quantitative biochemicalanalysis of GUS enzymic activity of nuclei isolated fromtransgenic tobacco plants revealed that one region is moreefficient than the other. The precise location of the NLSs wasdetermined using the onion transformation system. The firstNLS (NLS A) is located at the amino-terminal portion of the02 protein and has the structure of an SV40-type NLS (Fig.2, highlighted). The second NLS, an efficient NLS (NLS B),is located in the basic, DNA-binding domain and has abipartite structure (Fig. 2). Our analysis of 02 protein fusionsalso indicates that in general, amino-terminal fusions appearto be targeted to the nucleus more efficiently than carboxy-terminal fusions. This suggests that at least for the 02 NLSs,fusion to the carboxy-terminal of GUS may not permit properexposure and recognition by the import machinery. Futurestudies should examine whether one or both NLSs are nec-essary for nuclear targeting of the 02 protein.The finding that an efficient bipartite NLS is located in the

basic DNA-binding domain of 02 suggests that the NLSsidentified in two other b-ZIP tobacco proteins, TGA-1A and

TGA-1B (24), most likely also have a bipartite structure (seeFig. 2). It is tempting to speculate that the DNA-bindingdomains of other b-ZIP proteins may also function as NLSs(26). This would suggest a dual function for this domain: thecoordination of nuclear import, in addition to the acceptedrole in DNA binding.

NLSs of the R Protein

Genetic and structural evidence indicates that the maize Rgene encodes a nuclear transcriptional activating factor. Thisprotein contains a helix-loop-helix motif that is similar toregions of the transcriptional activators myo DI and myc (10).The R gene encodes a protein of 610 amino acids (69 kD),indicating that the R protein is imported into the nucleus byactive transport. To examine the nuclear localization of R,the reporter gene GUS has been fused to the R gene at amino-and carboxy-termini (R-GUS and GUS-R, respectively). Thefusions were then transiently expressed in onion epidermalcells (19). A histochemical analysis of onion epidermal cellsrevealed that proteins from either orientation of the GUS-Rand R-GUS fusions are localized in the nucleus.

Further analysis of chimeric constructs containing regionsof the R gene fused to the GUS cDNA revealed three specificNLSs that are capable of redirecting the GUS protein to thenucleus (Fig. 2). Amino-terminal NLS A (10 amino acids) israther unusual, as it has several arginines, and a similar NLSis found in only a few viral proteins (3). The medial NLS M(10 amino acids) is an SV40 type, and the carboxy-terminalNLS C is a MAT a2 type. NLS M and C are independentlysufficient to direct the GUS protein to the nucleus whenfused at the amino terminus of GUS. However, NLS A fusedto GUS, partitioned between the nucleus and cytoplasm.Similar partitioning is observed when all the NLSs are inde-pendently fused to the carboxy-terminal portion of GUS,indicating that the position of the NLS in the transportedprotein is important.

Experiments were also performed with fusions of the entireR and GUS to address the question of whether one, two, orall three NLSs are necessary for nuclear targeting of the Rprotein. A deletion analysis of the three NLSs indicates thatthe R-GUS and GUS-R protein fusions are redirected to thenucleus only when NLS A and M, or C and M, are bothpresent. In all other combinations-either a single NLS (A,M, or C) or double NLSs (A and C)-GUS fusions werelocated in both the nucleus and cytoplasm. These resultsindicate that multiple NLSs are necessary for nuclear target-ing of this protein. The fact that the R protein has twononhomologous signals that are necessary for its targeting tothe nucleus suggests that each sequence may be involved indifferent steps of nuclear transport or may interact withdifferent import components of similar function.

NUCLEAR TRANSPORT OF PLANT VIRAL PROTEINS

Many plant viruses encode proteins that accumulate indifferent compartments upon infection. Among the severalproteins involved in RNA replication of potyviruses are twonuclear localized proteins, NIa and NIb (15). Although thefunction(s) of these two proteins in the nucleus is not known,

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Plant Physiol. Vol. 100, 1992

the mechanism of their transport to the nucleus was recentlyaddressed. Both NIa and NIb were fused to GUS at theamino- and carboxy-terminal ends and transfected into to-bacco protoplasts. A subsequent analysis of GUS enzymicactivity demonstrated that carboxy-terminal fusion proteinsare enzymically more active than amino-terminal fusions,though either one can direct GUS to the nucleus. Systematicdeletions followed by fusions to the carboxy terminus of GUSwere performed on NIa in an attempt to identify sequencesthat confer nuclear localization of this protein. It was foundthat two short regions located within the amino-terminal partof NIa function as an NLS (Fig. 2, ref. 1). These regionsconsist of 11 and 30 amino acids, separated by 32 residues.

NUCLEAR IMPORT OF NUCLEIC ACIDS

The mechanism by which nucleic acids are transported tothe nucleus is largely a mystery. Agrobacterium is a plantpathogen that transfers DNA to most dicotyledonous plants.The T-complex of Agrobacterium consists of three compo-nents: a single-stranded DNA molecule, the T-strand, andtwo different proteins, one virD molecule and over 600 copiesof virE2 (2). The nopaline-type T-complex has a predictedlength of 3600 nm (2), which is approximately 60 timeslonger than the diameter of the nuclear pore. It has beenshown that most of the sequence of the T-DNA is notimportant; any DNA sequence located between the 25-bp T-DNA border repeats can be transported to the plant nucleusand fulfill its function (27). Therefore, it is proposed that theT-strand was most likely transported to the nucleus by itsassociated proteins. It was recently demonstrated that theVirD2 fused to GUS is localized to the nucleus when expressed

in tobacco plants and suspension cells (5, 7, 22). A bipartiteNLS (Fig. 2) at the carboxy terminus of VirD2 was identifiedas a result of detailed deletion analyses followed by fusionof these proteins to GUS (7). These results suggest that theVirD2 protein, which attaches to the 5' end of the T-strand,may act to direct the T-complex to the nucleus of the hostcell. However, recent work from the same laboratory indi-cates that another protein, VirE2, might also be involved innuclear transport of the T-complex.An analysis of a transiently expressed virE2 fused to GUS

showed that the VirE2 protein localized to the nucleus intobacco cells as well (2). Nuclear localization of VirE2 ismediated by two bipartite NLSs (NSE1 and NSE2) (Fig. 2),and an efficient nuclear localization of GUS can be achievedonly when both NLSs are present. It is suggested that VirE2serves as molecular chaperone by coating, unfolding, andtargeting the T-strand to the nucleus. These authors sug-gested that this phenomenon holds true for all ssDNA orRNA molecules that may be transported to the nucleus asunfolded nucleic acid-protein complexes (2, 11). Althoughthis is most likely true for import of T-strand and for someRNAs, there is strong evidence suggesting that some RNAspossess their own NLS (4). It was demonstrated that them3GpppN cap of U1-U5 snRNAs is a distinct NLS. However,the involvement of one or more proteins is also indicated.

CONCLUSIONS AND FUTURE PROSPECTS

In summary, several types of NLSs have been identified inplants. The SV40 type is recognized by the plant machineryand is sufficient to redirect a reporter protein to the nucleus;however, it is not always necessary for the targeting of

-NPC

NLS(s)-dependentNEM sensitive

C ATP-dependentATPYS sensitiveApyrase sensitiveWGA sensitiveTemperature sensitive

Figure 3. Nuclear import is a two-step process.

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TRANSPORT OF PROTEINS TO THE NUCLEUS

nuclear proteins. Multiple nonhomologous signals play a rolein the nuclear targeting of some nuclear proteins. The mostprevalent is a bipartite NLS, based upon the small amountof available data on plant nuclear proteins. It is obvious thatthe position of the NLS(s) and its availability to the importmachinery are very important as well.Although information is available about the different types

of NLSs, little is known about the molecular characteristicsof the translocation machinery. Generally, it is known thatin animal and yeast systems, import through the NPC is atwo-step process (Fig. 3). The first is an NLS-binding stepthat is sensitive to NEM (14). The second step is sensitive totemperature and wheat germ agglutinin and is ATP depend-ent (ATP-yS and apyrase sensitive) (3, 20). Experimentalevidence also indicates the existence of NBPs that are in-volved in nuclear localization of proteins containing an NLS(20). All of the NBPs that have been described to date are ofhuman, rat, or yeast origin, and the degree of sequenceconservation is uncertain, because only yeast and rat NBPsequences have been reported (9, 12).Although little has been learned of the identification of

NBPs originating in plants, some tantalizing hints recentlysurfaced. Stochaj and Silver (21) have reported the presenceof a protein in maize that is antigenically related to a 70-kDNBP from yeast nuclei. Recent, exciting work from JamesCarrington's laboratory also provides some clues about cel-lular factors that influence nuclear import in plants (16).

CYTOPLASM

Earlier, these authors showed that the potyviral protein NIaundergoes autoproteolytic cleavage at the amino terminusand releases a 6-kD protein. The initial experiments to ana-lyze the effect of proteolysis on nuclear transport of NIaindicate that transport to the nucleus was abolished whenthe cleavage site was inhibited, resulting in unprocessed NIain the cytoplasm. These results suggest that the release of the6-kD protein is required for nuclear localization of NIa, andimply an interaction of the NIa NLS, a 6-kD protein, andcytoplasmic protein factors.As discussed above, there are different types of NLSs that

have been identified in plants. Do these different NLSsrecognize a wide range of NBPs or is there a secondarystructural motif that is recognized by only a few NBPs? Someof these NBPs could play a role as cytoplasmic anchor,cytoplasmic receptor, or pore receptor, providing a way toregulate nuclear import (Fig. 4). Are there mechanisms thatcontrol the activity of NBPs during plant development?Plants, which must be more adaptive than animals to endureenvironmental changes, would benefit substantially fromsuch a level of regulation. The binding of NLSs to NBPs mayoccur in the cytoplasm or at the NPC. The cytoplasmic NBPsmay function as adaptor molecules between the transportedproteins and the actual import machinery of the NPC (Fig.4). Some of these NBPs may be developmentally regulated,and the NLS-binding activity of NBPs may be altered byphosphorylation (21). With the identification of several types

NPC{-

CA?_NUCLEUS

PPR?

t

Us.7

I M M40

CR ?Figure 4. Possible NLS recognition mechanisms.

CR ?

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Plant Physiol. Vol. 100, 1992

of NLSs in plants, all of these questions remain open forinvestigation.

ACKNOWLEDGMENTS

I would like to thank all members of the nuclear targeting group

in my laboratory: Marguerite Varagona, Glenn Hicks, Mark Shieh,and Antje Heese for their suggestions and exciting discussions thatstimulated my writing of this paper.

LITERATURE CITED

1. Carrington JC, Freed DD, Leinicke AJ (1991) Bipartite signalsequence mediates nuclear translocation of the plant potyviralNla protein. Plant Cell 3: 953-962

2. Citovsky V, Zupan J, Warnick D, Zambryski P (1992) Nuclearlocalization of Agrobacterium VirE2 protein in plant cells. Sci-ence 256: 1802-1804

3. Garcia-Bustos J, Heitman J, Hall MN (1991) Nuclear proteinlocalization. Biochim Biophys Acta 1071: 83-101

4. Goldfarb D, Michaud N (1991) Pathways for the nuclear trans-port of proteins and RNAs. Trends Cell Biol 1: 20-24

5. Herrera-Estrella A, Van Montagu M, Wang K (1990) A bacte-rial peptide acting as a plant nuclear targeting signal: theamino-terminal portion of Agrobacterium VirD2 protein directsa 13-galactosidase fusion protein into tobacco nuclei. Proc NatlAcad Sci USA 87: 9534-9537

6. Hinshaw JE, Carragher BO, Milligan RA (1992) Architectureand design of the nuclear pore complex. Cell 69: 1133-1141

7. Howard EA, Zupan JR, Citovsky V, Zambryski PC (1992) TheVirD2 protein of A. tumefaciens contains a C-terminal bipartitenuclear localization signal: implications for nuclear uptake ofDNA in plant cells. Cell 68: 109-118

8. Lassner MW, Jones A, Daubert S, Comai L (1991) Targeting ofT7 RNA polymerase to tobacco nuclei mediated by an SV40nuclear location signal. Plant Mol Biol 17: 229-234

9. Lee W-C, Xue Z, Melese T (1991) The NSRI gene encodes a

protein that specifically binds nuclear localization sequencesand has two RNA recognition motifs. J Cell Biol 113: 1-12

10. Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc,a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar totranscriptional activators and contains the myc-homology re-

gion. Proc Natl Acad Sci USA 86: 7092-709611. Mehlin H, Daneholt B, Skoglund U (1992) Translocation of a

specific premessenger ribonucleoprotein particle through thenuclear pore studied with electron microscope tomography.Cell 69: 605-613

12. Meier UT, Blobel, G (1992) Nopp 140 shuttles on tracks be-tween nucleolus and cytoplasm. Cell 70: 127-138

13. Moreland RB, Langevin GL, Singer RH, Garcea RL, HerefordLM (1987) Amino acid sequences that determine the nuclearlocalization of yeast histone 2B. Mol Cell Biol 7: 4048-4057

14. Newmeyer, DD, Forbes DJ (1990) An N-ethylmaleimide-sen-sitive cytosolic factor necessary for nuclear protein import:Requirement in signal-mediated binding to the nuclear pore. JCell Biol 110: 547-557

15. Restrepo MA, Freed DD, Carrington JC (1990) Nuclear trans-port of plant potyviral proteins. Plant Cell 2: 987-998

16. Restrepo-Hartwig MA, Carrington JC (1992) Regulation ofnuclear transport of a plant potyvirus protein by autoproteo-lysis. J Virol 66: 5662-5666

17. Robbins J, Dilworth SM, Laskey RA, Dingwall C (1991) Twointerdependent basic domains in nucleoplasmin nuclear tar-geting sequence: Identification of a class of bipartite nucleartargeting sequence. Cell 64: 615-623

18. Schmidt RJ, Ketudat M, Aukerman MJ, Hoschek G (1992)Opaque-2 is a transcriptional activator that recognizes a spe-cific target site in 22-kD zein genes. Plant Cell 4: 689-700

19. Deleted in proof20. Silver PA (1991) How proteins enter the nucleus. Cell 64:

489-49721. Stochaj U, Silver P (1992) A conserved phosphoprotein that

specifically binds nuclear localization sequences is involved innuclear import. J Cell Biol 117: 473-482

22. Tinland B, Koukolikova-Nicola Z, Hall MN, Hohn B (1992)The T-DNA linked VirD2 protein contains two distinct func-tional nuclear localization signals. Proc Natl Acad Sci USA 89:7442-7446

23. Tsuneoka M, Imamoto NS, Uchida T (1986) Monoclonal anti-body against non-histone chromosomal protein high mobilitygroup 1 co-migrates with high mobility group 1 into thenucleus. J Biol Chem 261: 1829-1834

24. van der Krol AR, Chua N-H (1991) The basic domain of plantB-ZIP proteins facilitates import of a reporter protein intoplant nuclei. Plant Cell 3: 667-675

25. Varagona MJ, Schmidt RJ, Raikhel NV (1991) Monocot regu-latory protein Opaque-2 is localized in the nucleus of maizeendosperm and transformed tobacco plants. Plant Cell 3:105-113

26. Varagona MJ, Schmidt RJ, Raikhel NV (1992) Nuclear local-ization signal(s) required for nuclear targeting of the maizeregulatory protein, Opaque-2. Plant Cell 4: 1213-1227

27. Zambryski P (1992) Chronicles from the Agrobacterium-plantcell DNA transfer story. Annu Rev Plant Physiol Plant MolBiol 43: 465-490

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