Proc. Natl. Acad. Sci. USAVol. 93, pp. 3853-3858, April 1996Plant Biology

Organ-specific and Agamous-regulated expression andglycosylation of a pollen tube growth-promoting protein

(pistil-specific gene expression/posttranslational modifications/proline-rich proteins)

ALICE Y. CHEUNG*, XIAO-YAN ZHANt, HONG WANG, AND HEN-MING WU

Department of Biology, Yale University, P.O. Box 208104, New Haven, CT 06520-8104

Communicated by Lawrence Bogorad, Harvard University, Cambridge, MA, January 2, 1996 (received for review November 9, 1995)

ABSTRACT Transmitting tissue-specific (TTS) protein isa pollen tube growth-promoting and -attracting glycoproteinlocated in the stylar transmitting tissue extracellular matrixof the pistil of tobacco. The TTS protein backbones have adeduced molecular mass of about 28 kDa, whereas the glyco-sylated stylar TTS proteins have apparent molecular massesranging between 50 and 100 kDa. TTS mRNAs and proteinsare ectopically produced in transgenic tobacco plants thatexpress either a cauliflower mosaic virus (CaMV) 35S pro-moter-TTS2 transgene or a CaMV 35S-promoter-NAGI(NAGI = Nicotiana tabacum Agamous gene) transgene. How-ever, the patterns of TTS mRNA and protein accumulationand the quality of the TTS proteins produced are different inthese two types of transgenic plants. In 35S-TTS transgenicplants, TTS mRNAs and proteins accumulate constitutively invegetative and floral tissues. However, the ectopically ex-pressed TTS proteins in these transgenic plants accumulateas underglycosylated protein species with apparent molecularmasses between 30 and 50 kDa. This indicates that thecapacity to produce highly glycosylated TTS proteins is re-stricted to the stylar transmitting tissue. In 35S-NAG trans-genic plants, NAG1 mRNAs accumulate constitutively invegetative and floral tissues, and TTS mRNAs are induced inthe sepals of these plants. Moreover, highly glycosylated TTSproteins in the 50- to 100-kDa molecular mass range accu-mulate in the sepals of these transgenic 35S-NAG plants.These results show that the tobacco NAGI gene, together withother yet unidentified regulatory factors, control the expres-sion of TTS genes and the cellular capacity to glycosylate TTSproteins, which are normally expressed very late in the pistildevelopmental pathway and function in the final stage offloral development. The sepals in the transgenic 35S-NAGplants also support efficient pollen germination and tubegrowth, similar to what normally occurs in the pistil, and thisability correlates with the accumulation of the highest levelsof the 50- to 100-kDa glycosylated TTS proteins.

A flower is a composite structure with four consecutive whorlsof organs, sepals, petals, stamens, and the pistil. The outerwhorls, sepals and petals, are most similar to leaves. The innerwhorls, the stamens and the pistil, are the male and femalereproductive organs, respectively. They are more complexorgans made up of multiple structural parts with specializedfunctions in sexual reproduction. The pistil has an apicallylocated pollen-receptive stigma, which is connected to thedistally located ovary by a stalk-like structure known as thestyle. The papillar and the secretory cells of the stigma and thestylar transmitting tissue cells secrete large amounts of mate-rials to their extracellular matrix. These together provide ahighly enriched environment for pollen germination and tubegrowth (1, 2), which occur at the terminal stage of floraldevelopment. The ability to support pollen germination and

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3853

tube growth is usually confined to these pistil tissues and to theovary. Other surfaces (e.g., the surfaces ofvegetative and othermature floral organs) do not support these processes. How-ever, this restriction may be relaxed by mutations (3) andappears not to be fully attained in young developing floraltissues in Arabidopsis (4).

Floral organ development is controlled by several majorregulatory genes that govern meristem and floral organ iden-tities. These genes in turn positively or negatively regulateprograms of gene expression, leading to the formation ofindividual components of the flower (see, for reviews, refs. 5and 6). Stigma and stylar transmitting tissue-specific geneshave been reported in a number of plants (e.g., refs. 7-12).However, how the expression of these genes is regulated by thefloral organ identity genes remain to be elucidated. Further-more, mechanisms behind the posttranslational modificationsof these gene products (e.g., the glycosylation of proteins inpistil tissues) are entirely unknown. It is possible that themaster regulatory genes for floral organ development definethe entire program of molecular and biochemical processesneeded to form these organs and maintain their functions. Oneof the best-characterized floral organ identity genes is Aga-mous, which is essential for normal stamen and pistil devel-opment (5, 6). Loss of the Agamous gene function results in theconversion of stamens into petals and the replacement of thepistil by an inner flower that reiterates the outer flower. Theseconversions give rise to reiterating whorls of sepals, petals,petals from the outside to the inside of the flower. TheAgamous gene from Arabidopsis (AG) (13) and its homologsfrom a number of other plants, including tobacco (NAG1),have been isolated and characterized (14-17). Ectopic expres-sion of AG, or its homologs from other species, leads tohomeotic changes in the floral organs in the transgenic plants(15, 17-21). Most noticeably, the sepals develop pistil prop-erties, and petals develop stamen properties. Presumably,ectopic expression ofAgamous genes in these nonreproductiveorgans is adequate to activate, directly or indirectly, theexpression of downstream regulatory and structural genes thatare necessary to confer pistil or stamen morphology.The tobacco stylar transmitting tissue-specific (TTS) glyco-

protein promotes pollen tube growth and attracts pollen tubesin vitro, and its sugar moieties are important for these biolog-ical activities (22-24). We show here that the ability toaccumulate highly glycosylated TTS protein is restricted to thestylar transmitting tissue and that the tobacco NAG1 geneparticipates in controlling the expression of TTS genes; it alsoregulates the glycosylation ofTTS proteins and confers ectopicpollen germination and tube growth.

Abbreviations: TTS, transmitting tissue specific; CaMV, cauliflowermosaic virus; WT, wild type.*To whom reprint requests should be addressed at: OML 454,Department of Biology, Yale University, P.O. Box 208104, NewHaven, CT 06520-8104.

tPh.D. candidate on leave of absence from Zhejiang AgriculturalUniversity, Hangzhou, China.

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MATERIAL AND METHODSPlant Material and Transformation. Sterile Nicotiana taba-

cum (SR1) plants grown in tissue culture were used in trans-formation experiments. Agrobacterium Ti plasmid-mediatedplant transformation was carried out according to describedprocedures (25). Transgenic and wild-type (WT) tobaccoplants were grown under greenhouse conditions.DNA Manipulation. The construction of the cauliflower

mosaic virus (CaMV) 35S promoter-TTS2 and CaMV 35Spromoter-NAG1 chimeric genes followed standard recombi-nant DNA methodology (26). The tobacco NAG1 gene wasobtained by PCR using oligonucleotide primers (synthesizedby the W. W. Keck Foundation Biotechnology Laboratory atYale University) flanking the 5' and 3' untranslated regions ofthe NAG1 gene (17). These two chimeric genes were intro-duced into the Agrobacterium Ti plasmid according to pub-lished procedures (25).RNA Gel Blot Analysis. RNA isolation and gel blot analysis

followed described procedures (22). Equal amounts of RNAwere loaded in each lane for all of these gels. DNA probes forthese blots were labeled with 32p by random-priming (26).

Protein Analysis. Protein isolation, immunoblot analysis,and immunohistochemical analysis followed described proce-dures (22), except that colloidal gold (5 nm)-labeled secondarygoat antibodies against rabbit immunoglobulins and silverenhancement (Amersham) were used for the immunohisto-chemical analysis. For protein blots, equal amounts of proteinswere loaded in each lane of the gels.

Protein Deglycosylation. Chemical deglycosylation by hy-drogen fluoride followed a published procedure (27). Enzy-matic deglycosylation by N-glycosidase F followed the manu-facturer's (New England Biolabs) recommended procedures.

Pollination Assays. Pollen from freshly dehisced WT an-thers were deposited on the sepals ofWT, transgenic 35S-TTSand transgenic 35S-NAG plants. They were maintained undergreenhouse conditions for 24 hr and treated for scanningelectron microscope observations (3).

RESULTSCaMV 35S-TTS2 Gene Induces Ectopic Accumulation of

Underglycosylated TTS Proteins. A chimeric CaMV 35S-TTS2 gene (Fig. la) was used to transform tobacco plants. Alarge majority of these transgenic plants (35S-TTS plants)appear normal in all vegetative and reproductive phenotypes.Contrary to the stylar-specific accumulation of TTS mRNAsand proteins in WT plants (9, 22), the phenotypically normal35S-TTS transgenic plants accumulate comparable levels ofTTS mRNAs (data not shown) and proteins in all vegetativeand floral tissues (Fig. 2a), consistent with the constitutivenature of the CaMV 35S promoter. However, the TTS proteinsin these 35S-TTS plants have apparent molecular massesranging between 30 and 50 kDa, except in the style, where 30-

aCaMV35S-TTS2

CaMV35Spromoter

bCaMV35S-NAG1

CaMV35Spromoter

TTS2cDNA

NAG1

polyA-site

polyA-site

FIG. 1. (a) Chimeric CaMV 35S-TTS2 gene. (b) Chimeric CaMV35S-NAG1 gene.

a<-----WT ----><---35S-TTS--->

L SePeSt SvOv LSePeSt SvOv

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FIG. 2. (a) Immunoblot analysis of TTS proteins in a WT and arepresentative transgenic 35S-TTS plant. The blot was reacted withTTS protein antibodies and alkaline phosphatase-labeled secondaryantibodies. L, leaf; Se, sepal; Pe, petal; St, stamen; Sy, stigma/style; Ov,ovary. The numbers on the right-hand side of this blot indicatemolecular masses in kDa. (b and c) Immunohistochemical detection ofTTS proteins in the style of a WT plant (b) and a representativetransgenic 35S-TTS plant (c). The sections were stained with TTSprotein antibodies and colloidal gold (5 nm)-labeled secondary anti-bodies and were silver-enhanced. TT, transmitting tissue; C, corticaltissue; V, vascular bundle; E, epidermis. Dark areas indicate golddeposits, revealing the presence of TTS proteins.to 100-kDa TTS protein species accumulate. These results indi-cate that organs other than the style do not accumulate highlyglycosylated TTS protein even when they synthesize its mRNAsand protein backbones. Immunohistochemical analysis of sec-tions across the styles of these transgenic 35S-TTS plants showedthat in addition to the transmitting tissue, the epidermis, cortical,and vascular tissues also accumulate TTS proteins (Fig. 2c).Together, these results suggest that the underglycosylated 30- to50-kDa TTS proteins in the styles of the transgenic 35S-TTSplants are produced by the nontransmitting tissues.CaMV 35S-NAGI Gene Induces Ectopic TTS Gene Expres-

sion in Transgenic Sepals. A chimeric CaMV 35S-NAG1 gene(Fig. lb) was used to transform tobacco plants. These trans-formed plants (35S-NAG plants) have anomalous floral phe-notypes similar to those reported previously (15, 17-21),including the transformation of sepals into structures withpistil properties. In the 35S-NAG transgenic plants with themost severe phenotypes, their sepals become highly elongated,and the apical region of these sepals assume a stigmatoidappearance (Fig. 3 b and d). In nontransgenic tobacco plants,NAG1 mRNAs are present in extremely low levels in thestamen, the stigma, and the style and are present in low levelsin the ovary of the mature flowers (Fig. 4a). In the transgenic35S-NAG plants, the NAG1 mRNAs accumulate to very highlevels in all the floral tissues (Fig. 4a) and in the leaves androots (data not shown). Interestingly, TTS mRNAs also be-come detectable in the sepal tissues of these transgenic plants.Furthermore, at least two other transmitting tissue-specificmRNAs, MG15 (7) and j-(1-3)-glucanase (28), are alsoinduced in these sepals (Fig. 4a). Although NAG1 mRNAs aredetected constitutively in all floral organs of the transgenic35S-NAG plants, the expression of TTS genes is not consti-tutive in the flower. Furthermore, the ectopic accumulation ofhigh levels of NAG1 mRNAs is not adequate to affect thespecificity of all floral organ-specific genes. For example, theaccumulation pattern of at least one stamen-specific generemains unchanged in these transgenic plants (Fig. 4a).

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a

0*wZi

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FIG. 3. Scanning electron microscopy of the sepals ofWT and transgenic 35S-NAG plants and their response to pollination. (a) Sepal tips froma WT plant. (Bar = 1 mm.) (b) Sepals from a transgenic 35S-NAG plant showing fused sepals with stigmatoid tips (arrows). (Bar = 1 mm.) (c)Pollen grains (Pg) on a WT sepal blade at 24 hr after pollen deposition on this surface. (Bar = 50 ,um.) Similar surface structure and pollen behaviorwere observed at the sepal tip. (d) The stigmatoid structures (arrows) of the sepals of a transgenic 35S-NAG plant where pollen grains weredeposited. (Bar = 1 mm.) (e) The surface of an unpollinated stigmatoid sepal tip of a 35S-NAG plant. Presence of exudates and a lack of surfacemorphology on these cells contrast the properties of WT sepal surface (compare with c). (Bar = 50 ,um.) (f) Pollen grains (Pg) on the blade ofa sepal of a transgenic 35S-NAG plant at 24 hr after pollen deposition. The highly elongated cells contrast the approximately isometric cells foundon the blade of nontransgenic sepals (C). T, trichome. (Bar = 50 ,um.) (g-i) The pollinated stigmatoid surfaces of a 35S-NAG plant (shown in d)at 24 hr after pollen deposition. Many pollen grains (Pg) germinated pollen tubes (pt), some of which penetrated the surface (arrows). Thesepollinated surfaces became covered with copious amounts of exudates. (Bars = 50 ,um.)

Expression of TTS Proteins in the Sepals of Transgenic35S-NAG Plants. Transgenic 35S-NAG plants also ectopicallyaccumulate TTS proteins in their sepals (Fig. 4b). These TTSproteins are predominantly between 50 and 100 kDa, similarto those found in the styles. Furthermore, they are also highlysoluble proteins secreted to the tissue surface and can bereleased from intact sepals by gentle, low-salt washes (data not

shown), similar to the TTS proteins found in the transmittingtissue (22, 23).

Immunohistochemical analysis of tissue sections from thesepals of the 35S-NAG transgenic plants showed that thelevels of TTS proteins are the highest in the apical region anddecline toward the lower parts of this organ (Fig. 5). Thiscontrasts with the relatively constant level of TTS proteins

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< WT----><-35S-NAG->a

Se Pe St SyOv Se PeSt SyOvb1 2 3 4 5 6 7 8 910

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Style sepal sepalNAG1

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#0 fT MG-15

P-glu- :,..

canase

BHF - + + - + +HF+E - - + - - +

FIG. 4. (a) RNA gel blot analysis for NAG1, TTS, MG-i5, 13-(1-3)-glucanase, and a stamen-specific mRNA (OB) in a WT and a transgenic35S-NAG plant. For NAG1 mRNA detection, only the 3' half of the NAGI gene (without the MADS box region) was used as a probe uniquefor the NAG1 mRNA. Se, sepal; Pe, petal; St, stamen; Sy, stigma/style; Ov, ovary. (b) Immunoblot analysis of TTS proteins in the WT sepals andovaries (lanes 3 and 4) and in the sepals (lanes 5, 7, and 9) and ovaries (lanes 6, 8, and 10) of three representative transgenic 35S-NAG plantsexpressing different levels of TTS proteins. Lane 1, TTS proteins from WT styles; lane 2, TTS proteins from WT styles after hydrogen fluoridedeglycosylation. (c) Immunoblot analysis of the glycosylation properties ofWT stylar TTS proteins and the sepal-expressed TTS proteins from the35S-TTS and the 35S-NAG transgenic plants. These proteins were either untreated, treated with hydrogen fluoride (HF), or treated consecutivelywith HF and N-glycosidase F (E) as indicated in the figure. The numbers on the right-hand side of the blots in b and c indicate molecular masses in kDa.

found throughout the sepals of the 35S-TTS transgenic plants.Tissue sections of the 35S-NAG transgenic sepals also re-vealed that they have a very compact cell arrangement more akinto that found in pistil tissues, in contrast to the relatively looselypacked cells in sepals ofWT and transgenic TTS-35S plants.

Ectopically Expressed TTS Proteins Are O-Glycosylated toDifferent Extents. Anhydrous hydrogen fluoride treatment ofglycoproteins cleaves all sugar linkages and, under severeconditions, 0-glycosidic linkages of amino sugars. Peptide

WT 3!

bonds, and N-glycosidic linkages between asparagine residuesand N-acetylglucosamine are left intact (29). N-glycosidase Fcleaves the innermost N-acetylglucosamine of oligosaccha-rides from asparagine residues ofglycoproteins (30, 31). NativeTTS proteins are reduced from their 50- to 100-kDa molecularmasses to 30-kDa protein species by hydrogen fluoride de-glycosylation. Further deglycosylation of the chemically de-glycosylated 30-kDa TTS proteins by N-glycosidase F reducesthem to about 28 kDa (Fig. 4c and ref. 22). Chemical degly-

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Mid-.e >XUL t~~sectioneRS

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FIG. 5. Immunohistochemical detection of TTS proteins in the sepals of a WT plant, a transgenic 35S-TTS plant, and a transgenic 35S-NAGplant. Sections from the tip, mid-section, and the base of the sepals are shown. The sections were treated with TTS antibodies and with gold-labeledsecondary antibodies and counterstained with fast green. Arrowheads point to gold deposits revealing the locations of TTS proteins.

--110

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cosylation of the underglycosylated TTS proteins from the 35S-TTS plants and of the 50- to 100-kDa TTS proteins from the35S-NAG transgenic plants reduces their apparent molecularmasses similarly to 30 kDa. Enzymatic deglycosylation of these30-kDa TTS proteins from both types of transgenic plantsreduces their molecular masses further toward 28 kDa (Fig. 4c).These results indicate that the TTS proteins produced ectopicallyin the 35S-TTS and 35S-NAG transgenic plants have signifi-cantly different levels of O-linked glycosylation but are compa-rably glycosylated at their N-linked glycosylation site(s).The Sepals of Transgenic 35S-NAG Plants Support Pollen

Germination and Tube Growth. The apical region of the sepalsof the 35S-NAG transgenic plants is covered with exudates,contrary to the relatively exudate-free wild-type sepals (com-pare Fig. 3 e and c). Tobacco pollen grains applied to the sepalsof the 35S-NAG transgenic plants germinate efficiently in theapical region, and their tubes elongate considerable distances,often penetrating the sepal surface (Fig. 3 g-i). However,pollen grains do not germinate on the blade of these transgenicsepals, where cells are significantly longer than in the sepals ofcontrol plants (Fig. 3f). Pollen grains applied to WT sepals(Fig. 3c) or sepals from the 35S-TTS transgenic plants (datanot shown) also do not germinate.

DISCUSSIONTTS Protein Glycosylation. The stylar transmitting tissue of

angiosperms is highly enriched in glycosylated compounds,some of which are believed to be important for the pollinationprocess. Among these sugar-containing molecules are manyglycoproteins. These include many proline-rich proteins-e.g.,TTS proteins and other arabinose- and galactose-containingproteins (32, 33), which collectively belong to the arabinoga-lactan protein family (34-36)-and extensin-like proteins (6, 7,36). Therefore, the stylar transmitting tissue must have thecapacity for efficient protein glycosylation and for makingdiverse types of sugar linkages. The exclusive ability for thestylar transmitting tissue to fully glycosylate TTS proteins (Fig.2) indicates that some aspects of protein glycosylation areunique in this tissue.The inability of nontransmitting tissue to complete the

O-glycosylations of TTS proteins (Fig. 4 b and c) may be dueto the depletion of the needed enzymatic activities. However,this is probably not likely since all cell types produce manyglycosylated compounds, and the amounts of ectopically pro-duced TTS backbone polypeptides in the transgenic 35S-TTSplants are not excessively high. Furthermore, 35S-TTS plantsproducing very high or very low levels of TTS proteins showsimilar extent of TTS protein glycosylation levels (data notshown). Alternatively, the nonstylar tissues may be lackingenzymes for the posttranslational modifications unique to TTSproteins-e.g., to hydroxylate all the proline residues, toglycosylate certain hydroxyproline residues (37), or to com-plete one or more specific linkages between carbohydratemolecules, thus prohibiting further condensation to completethe glycosylation of TTS proteins. Complete characterizationof the sugar moieties and the sugar-peptide linkages on thestylar transmitting tissue-produced and the non-stylar tissue-produced TTS proteins in the transgenic 35S-TTS plants willbe necessary to distinguish among these possibilities.

Examples of cell type-specific carbohydrate epitopes arecommon to many biological systems. In plants, arabinogalac-tan proteins are ubiquitous to all cell and tissue types and inall angiosperms that have been examined (34-36). They arelargely extracellular or plasma membrane-associated proteinsand are believed to carry out diverse functions that involveinteractions on the cell surface. The specificity of these inter-actions inevitably falls upon the differences in the polypeptidebackbones and their sugar modifications. The characterizationof cDNAs for several arabinogalactan proteins reveals signif-

icant heterogeneity in their polypeptide sequences (9, 33, 38,39). It has also been shown that several sugar epitopes displaydevelopmental stage- or cell type-specific expression patterns(40-43). Differential sugar epitope expression patterns mayhave resulted from differences in the expression of genesencoding glycoproteins (see ref. 36), from differential post-translational modifications of proteins as reported here, orfrom mechanisms differentially regulating the synthesis ofsugar molecules. In plants, the Golgi apparatus is the site forthe synthesis and processing of complex polysaccharides andglycoproteins. Golgi stacks with different and distinct mor-phological and chemical staining properties have been ob-served in the different tissue types of the tobacco and Arabi-dopsis root tips (44). This, together with the observations oftissue-differential expression of sugar epitopes, led to thesuggestion that during plant development, the Golgi apparatusis tailored in a tissue-specific manner to meet the syntheticneeds of glycosylated macromolecules in each tissue (44). Itwill be interesting to examine whether unique structural orchemical properties are associated with the Golgi bodies in thestylar transmitting tissue, which may be related to its uniqueglycosylation ability.TTS proteins interact intimately with pollen tubes in vivo

and in vitro to mediate its activities in pollen tube growthpromotion and attraction (23, 24). It has been shown that thesugar moieties on TTS proteins are important for their activ-ities and that pollen tubes can deglycosylate these proteins.Furthermore, TTS proteins display a gradient of increasingglycosylation from the stigmatic end to the ovarian end of thestyle, the same direction as pollen tube growth. This TTSprotein-bound sugar gradient has been speculated to have arole in the directional growth of pollen tubes within the style.The transmitting tissue-specific glycosylation of these proteinsreported here is another intriguing property associated withthe sugar moieties on TTS proteins. It is possible that theoligosaccharides on TTS proteins have specific functionalsignificance. Detailed structural-functional analysis of thesugar moieties on TTS proteins will be needed to furtherunderstand their significance.TTS Gene Expression Is Controlled by an Agamous-

Regulated Gene Expression Pathway. Agamous is a MADSbox protein and a key regulator for stamen and pistil devel-opment (5, 6, 45). The proteins encoded by theArabidopsis andthe maize Agamous genes have been shown to directly regulateanother Arabidopsis MADS box protein gene,AGL5, which isexpressed temporally afterAG expression is initiated but stillrelatively early in floral development (16, 46). In the sepals ofthe transgenic 35S-NAG plants, a regulatory cascade must beset up by the expression of NAG1. This leads to the expressionof a battery of genes, including those that are expressed verylate in floral developmental time frame, such as TTS, MG15,and 3-(l-3)-glucanase, which participate in conferring eitherpistil structural properties or the ultimate pistil functions inplant reproduction. NAG1 apparently also activates, directly orindirectly, the activities that posttranslationally glycosylateTTS protein backbones.The accumulation of TTS mRNAs and proteins in the sepals

of 35S-NAG transgenic plants (Fig. 4) suggests that the NAG1protein, either together with other regulatory factors alreadypresent in the sepals or together with factors that it induces inthe sepals, activates the TTS genes. However, it is clear thatNAG1 alone is inadequate to accomplish TTS gene activation,since none of the other nonstylar tissues in the transgenic35S-NAG plants expresses TTS genes despite high levels ofexpression from the CaMV 35S-NAG1 transgene (Fig. 4a).This is evident even in the ovary of the WT flowers where,despite a detectable level of NAG1 mRNA (Fig. 4a), TTSmRNAs and proteins do not accumulate to detectable levels(9, 22). The presence of very high levels of NAG1 mRNAs inthe ovaries of the transgenic 35S-NAG plants cannot over-

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come the mechanism to suppress TTS gene expression there(Fig. 4a). Furthermore, normal expression of NAG1 in theovary of 35S-TTS transgenic plants (data not shown) is notassociated with full glycosylation of the CaMV 35S promoter-expressed TTS protein backbones there (Fig. 2a). Therefore,multiple regulatory mechanisms must exist to exclude fullyglycosylated TTS proteins from the ovary. Either factors thatrepress TTS gene expression are present in the ovary (andother nonstylar tissues), or additional factors are needed to acttogether with the NAG1 protein, orNAG1-activated factors, toactivate TTS gene expression there. Several important regu-latory protein-binding sites have been located in the 5'-upstream region of a TTS gene, and these include at least twotarget sites for MADS box proteins (H.-m.W., X.-y.Z., andA.Y.C., unpublished results). Whether, and how, the NAG1protein, other Agamous-like proteins, or other novel transcrip-tion factors induced by NAG1 interact with the TTS generegulatory region need to be investigated.Long-Range Molecular and Biochemical Effect of Agamous

on the Pistil. The conversion of sepals in the 35S-NAGtransgenic plants to pistillate structures capable of supportingpollen germination and tube growth (Fig. 3) must have re-sulted from the expression of at least a majority of the genesnecessary for normal pistil formation and functions. Thefunctional role of Agamous in floral organ identity is clearlydemonstrated by mutant analysis and supported by the specificspatial expression pattern of the Agamous gene early in floraldevelopment. However, Agamous mRNAs continue to accu-mulate in restricted cell types late in normal floral develop-ment, including in the pistil. The functional importance ofAgamous in late floral development has not been elucidatedbecause the absence of the pistil in Agamous mutants pre-cludes the analysis of properties based on late Agamous geneexpression (see ref. 45). The NAG1-induced sepal expressionof pistil-specific genes, which function relatively late in devel-opmental time frames, and the sepal ability to fully glycosylateTTS proteins and to support pollen germination and tubegrowth are molecular, biochemical and cellular evidences forthe long-range effect that NAG1 has on late pistil developmentand functions in tobacco. Therefore, it is probable that at leastone of the functions of Agamous in late floral developmentalstages is to maintain the expression of genes or the activity oftheir gene products, which are necessary for housekeeping orspecific biological processes that occur in the pistil.We showed previously the Arabidopsis mutant fiddlehead

promiscuously supports pollen germination and tube growthover its entire aerial epidermis. It was suggested that thefiddlehead mutation results in the ectopic expression of a pistildevelopmental program that confers properties normallyunique to the female reproductive system (3, 47). The induc-tion of pistil morphological, molecular, and biochemical prop-erties in the sepals of the transgenic 35S-NAG plants isconsistent with the mechanism proposed behind the action ofthe fiddlehead mutation. However, the promiscuous nature ofthe fiddlehead mutant phenotype suggests that this mutationmost probably results in the relaxation of mechanisms thatsuppress pistil properties in nonpistil tissues. The sepals of thetransgenic 35S-NAG plants and the fiddlehead plants will beuseful materials for the identification of female factors con-tributing to pollen germination and tube growth.We thank Mrs. Nancy Carrignan for her skillful and patient assis-

tance in the preparation of this manuscript. 3-(1-3)-glucanase cDNAclones were generous gifts from Dr. Robert Fluhr (Weissman Institute,Israel). We thank Dr. Michael Hahn for communicating results onArabidopsis root cell-specific accumulation of AGP epitopes prior topublication. X-y.Z. and H.W. were predoctoral fellows supported byThe Rockefeller Foundation. This work was supported by grants fromthe U.S. Department of Agriculture (9303024) and the NationalInstitutes of Health (GM52953).

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