lmmunological detection of potential signal … · the putative role of the 29 ... chromosomes,...

Plant Physiol. (1997) 113: 801-807

lmmunological Detection of Potential Signal-Transduction Proteins Expressed during Wheat Somatic Tissue Culture

Aimé Nato*, Ali Mirshahi, Gabrielle Tichtinsky, Massoud Mirshahi, Jean-Pierre Faure, Danièle Lavergne, Jacques D e Buyser, Cécile Jean, Georges Ducreux, and Yves Henry

Laboratoire de Morphogénèse Expérimentale Végétale, Bâtiment 360, Université Paris XI, 91 405 Orsay, France (A.N., A.M., D.L., J.D.B., C.J., G.D.); Centre de Recherches Biomédicales des Cordeliers, lnstitut National de Ia

Santé et de Ia Recherche Médicale U86, 15 Rue de I’Ecole de Médecine, 75270 Paris 06, France (M.M., J.-P.F.); and Laboratoire de Biologie du Développement des Plantes, Unité de Recherche Associée Centre National de Ia

Recherche Scientifique 11 28, IBP, Bâtiment 630, Université Paris XI, 91405 Orsay, France (G.T., Y.H.)

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (C-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to C-protein (a subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-Gcu antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multi- plication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrestin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.

The development of a multicellular embryo from a single egg cell requires both the determination and the organization of many cell types (Mayer et al., 1991; Meinke, 1995). In plants either individual somatic or gametic cells can give rise to true embryos through in vitro culture. In CS wheat (Triticum aestivum) about 95% of the immature embryos cultured on a simple medium produce embryogenic cultures (De Buyser et al., 1992). Visual observation and his- tology confirm that the compact, yellowish embryogenic cultures possessed embryos and green areas at their surface and unorganized parts (i.e. callus) in connection with the culture medium. The nonembryogenic calli are soft and

the organized parts for subculture (De Buyser et al., 1988). Using 42 aneuploid lines of the embryogenic variety CS, we have previously demonstrated that at least one major gene on wheat chromosome arm 3DL controls the somatic embryogenesis capacity (Henry et al., 1994). These aneuploid lines, which lack chromosomes or chromosome arms, were used to investigate the chromosomal localization of the gene encoding an arrestin-like protein during somatic embryogenesis.

G-proteins are members of a superfamily of proteins that bind and hydrolyze guanine nucleotide (Rens-Domiano and Hamm, 1995). Among them, signal-transducing G-proteins are classified into two groups. One is the heterotrimeric large G-proteins, consisting of a, p, and y subunits, coupled to receptors of the seven-transmembrane- segment class. A few plant G-protein a-subunit genes (proteins of about 45 kD) have been isolated, including GPAl from Arabidopsis, homologs of GPAla from lotus and maize, TGAl from tomato (for review, see Ma, 1994), RGAl from rice (Ishikawa et al., 1995; Seo et al., 1995), and SGAl from soybean (Kim et al., 1995). The second group consists of the low-molecular-mass (21-36 kD) monomeric G-proteins (Ma, 1994). Since the early 1990s, molecular approaches have identified more than 60 small G-proteins in plants (Ma, 1994). Studies have shown that in higher plants, G-proteins are involved in hormonal and light sig- na1 transduction (Bowler and Chua, 1994), in the plant defense response (Legendre et al., 1992), and in the regulation of ion channel activities (Li and Assmann, 1993).

The arrestins belong to a family of monomeric soluble proteins conserved among animal species (for review, see Palczewski, 1994; Craft and Whitmore, 1995). The primary structure and antigenicity of arrestins were well conserved during animal evolution. Recent studies suggest the exis- tente of at least four arrestins in mammals. Retina1 arrestin is a soluble 48-kD protein that regulates phototransduction ~-

translucent without any differentiated structure (Henry and De Buyser, 1997). The capacity for somatic embryogenesis was maintained over a long period of time by selecting

* Corresponding author; e-mail aime.nat0Qmve.u-psud.fr; fax

Abbreviations: CS, cv Chinese Spring; DS and DL, short (S) and long (L) chromosome arms; DT and NT, aneuploids (ditelosomic and nullisomic-tetrasomic); G-protein, guanine nucleotide-binding protein; LHCPII, light-harvesting chlorophyll a / b-binding complex; mAb, monoclonal antibody; NDPK, nucleoside diphosphate kinase. 33-1- 69 - 85-54 -90.

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Nato et al. Plant Physiol. Vol. 11 3 , 1997 802

in retinal rods. In rod outer segments arrestin binding to photoactivated, phosphorylated rhodopsin (Kiihn, 1978) inhibits rhodopsin-Ga-protein interaction and thereby ter- minates the phototransduction process (Wilden et al., 1986). Similarly, p-arrestins protect the phosphorylated P-adrenergic receptors from binding to Ga-protein (Lohse et al., 1990). Arrestins have been immunodetected in photoreceptor cells (Mirshahi et al., 1985), nonphotosensitive animal cells (Mirshahi et al., 1989), and various systems, including yeast (Jeansonne et al., 1991). Proteins recog- nized on immunoblots by mAbs against retinal arrestin were also found in soluble tobacco cell extracts and in isolated thylakoid membranes from tobacco chloroplasts and Chlamydomonas (Mirshahi et al., 1991, 1992).

NDPK (EC.2.7.4.6.) plays a key role in controlling the metabolic flow of phosphate groups among nucleoside di- and triphosphates. The supply of trinucleotides for protein synthesis and cytoskeleton building may be important for embryogenesis (Yano et al., 1995). The enzyme from eukaryotes is a hexamer of identical subunits with a particu- lar mononucleotide binding fold (Dumas et al., 1992). A striking homology of the amino acid sequence of spinach NDPK with both the human and Drosophila melanogaster NDPKs was established (Nomura et al., 1992; Moisyadi et al., 1994). This led to the hypothesis of a possible contribu- tion of the NDPK protein to the development of plant tissue, including cell proliferation, through signal- transduction pathways (Wallet at al., 1990).

In vitro wheat somatic embryogenesis, initiated from immature zygotic embryo culture, was used to test the involvement of NDPK-, G-protein-, and arrestin-like proteins. We developed an immunological approach using mAbs and polyclonal antibodies raised against defined sequences of the animal proteins. In this study we demon- strate that molecules immunoreactive with antibodies to Ga-protein, arrestin, and NDPK are expressed during wheat tissue culture, and that one arrestin-related protein was specifically associated with somatic embryogenetic potential. The possible involvement of these proteins in hor- mona1 or light signal-transduction pathways is discussed.

MATERIALS AND METHODS

The hexaploid wheat (Triticum aestivum L., 2n = 6x = 42) CS was used as a reference due to its high performances in somatic tissue culture (De Buyser et al., 1992). CS DT and NT lines were provided by S.M. Reader (Agricultura1 and Food Research Council, Cambridge Laboratory, UK). The 36 DT lines were designated by their homologous chromosome group (1-7), genome (A, B, or D), and chromosome arm length (L = long, S = short). Each DT line lacks one pair of homologous chromosome arms. For example, the CS DT3DS line possesses the short arm (S) but lacks the long arm (L) of chromosome 3 of the D genome, which allows the effect of the deficient long arm to be evaluated. Each of the 6 NT lines lacks a given pair of homologous chromosomes, which is compensated for by an extra pair of one of their homolologous chromosomes. For example, the N2DT2A line possesses four 2A homologous chromosomes but lacks the chromosome pair 2D. The effect of the chro-

mosome arms 4AL and 5AL was not studied. This material and the plant culture conditions have been previously described (Henry et al., 1994).

Tissue Culture

Fourteen days after anthesis the CS, CS DT, and CS NT immature seeds were harvested and surface-sterilized (Henry et al., 1994). The immature embryos were excised and cultured on a medium containing Murashige and Skoog (1962) inorganic salts and vitamins, 20 g L-' SUC, 2 mg L-l 2,4-D, and 6.5 g L-l agarose, with the pH adjusted to 5.8 before autoclaving. Embryogenic and nonembryogenic CS cultures were established by a visual selection at each subculture (De Buyser et al., 1988, 1992). A11 of the in vitro cultures were performed in the same culture room at day/night temperatures of 27 ? 1°C and 24 2 1°C under low illumination (10 p E m-* s-l) with a 16-h photoperiod (Henry et al., 1994).

Antibodies

The following antibodies were used for immunoblotting procedures.

(a) A rabbit immune serum was prepared against a synthetic oligopeptide (GAGESGKSTIVKQM) representing a highly conserved region of mammalian heterotrimeric G-protein a! subunits. This region corresponds to a domain involved in the binding of GTP. It forms a loop that bonds with the a and p phosphates of GTP or GDP (Bourne et al., 1991). The sequence is also highly conserved in plant Ga- proteins, and displays similarities with animal, yeast, and plant small G-proteins (Fig. 1).

G A G E S G K S T I V K QM synthetic oligopeptide

Gs, bovine

Go, rat

- - - - - - - _ - _ _ - . - - _ _ - - - _ _ _ _ _ - _ _

- - - - - - - - - - - . E -

- _ _ - - - _ _ _ - F - - I - - - D - - _ _ _ _ F . - I

- - _ - - - - _ - - F - - I

- - - G V - - - A L T I Q o - D - G T - - T - F - - R H - D - G T - - T - F - - R H - D - G T - - T - F - - R H - D - G T - - T - F - - R H - D - G T - - I - F - - R H - D - G T - - T - F - - R L

Gi, mouse

GTI rods, bovine

GT2 cones, bovine

GPAI, Arabidopsis

TGA 1, tomato

RGA1, rice

c-Ha-ras, human

Ran, human

Ran GSPI, yeast

Ran, tomato

Ran, Arabidopsis

Ran,Vicia faba

Ran, tobacco

-: identity; o: gap

Figure 1. Sequence homology of the phosphate-binding regions between a synthetic oligopeptide and Gol subunits or small CTP- binding proteins.

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lmmunological Detection of Potential Signaling Elements 803

(b) Four mAbs against bovine retina1 arrestin (Faure et al., 1984; M. Mirshahi, unpublished data) were selected for this study. mAbs SlA3, S9E2, M4H10, and S8D8, respectively, recognize epitopes in the C-terminal (amino acids 371-380, QDENFVFEEF, and 361-368, EDPDTAKE), intermediate (296-301, DGKIKH), and N-terminal (40-50, PVDGVVLVDPE) regions of the 404 residues of the amino acid sequence of bovine arrestin. A mixture of these four mAbs was used. The epitopes for S8D8, M4H10, and S1A3 are highly conserved in all mammalian arrestins.

(c) The 143-2 anti-NDPK mAb (a gift from F. Traincard, Institut Pasteur, Paris) was obtained following immuniza- tion of a Biozzi mouse strain with human NDPK-A (i.e. nm23 H1) that was extracted from erythrocyte and purified as described by Presecan et al. (1989). This mAb detects the human NDPK-A protein by ELISA and western blotting and is also reactive with human NDPK-B, Dictyostelium discoideum NDPK, and Drosophila NDPK, but does not recognize Escherichia coli or Myxococcus xanthus NDPKs (M. Veron and F. Traincard, personal communication). The 143-2 mAb recognizes the epitope PADSKPGTIRGDF- CIQV, which contains the highly conserved sequence PGTIRGDF.

(d) An antiserum against Chlamydomonas-purified LHC- PII (Dubertret et al., 1994) was obtained from Lewis rats immunized by a single foot-pad injection (100 pg of protein mixed with Freund's adjuvant).

Protein Extraction

We used both wheat immature embryos and in vitro cultures obtained 8 weeks after subculture. The wheat immature embryos and callus tissues were ground in a cold mortar and then homogenized in an extraction buffer containing 50 mM Tris-HCI, pH 8.0, 10 mM MgCl,, 10 mM DTT, and 0.2 mM PMSF. The homogenates were ultracentrifuged at 120,OOOg for 30 min and the supernatant was taken as a soluble protein extract. The membrane pellet was washed twice by resuspension in the extraction buffer containing 0.7 M NaCl (1 h, 4"C), followed by centrifugation at 120,000g for 20 min. The final pellet was resuspended in the extraction buffer and treated with 1% (v/v) 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate for 1 h at room temperature. The protein content was determined as described by Sedmak and Grossberg (1977).

PACE and lmmunoblotting

SDS-PAGE was performed according to Laemmli (1970). For each sample tested, 30 pg of denatured protein was

electrophoresed for 2 h in a 15% polyacrylamide gel. The proteins were electrotransferred to PVDF membranes for 1 h using a semidry apparatus (Pharmacia LKB Multiphor 11). The membranes were incubated with the mouse mAbs anti-arrestin or anti-NDPK, or with the antiserum against the Ga peptide. Antigen-antibody complexes were detected with sheep anti-mouse or anti-rabbit biotinylated antibodies, followed by an incubation with streptavidin- biotinylated horseradish peroxidase complex (Amersham). 4-Chloro-l-naphthol-H,O, was used as the substrate reaction mixture. The specificity of the immune reaction was assessed by omitting the specific antibody or by incubating membranes with the preimmune serum.

RESULTS

lmmature Embryo Analysis

The wheat somatic tissue cultures were initiated from zygotic immature embryos, which were used here as a reference. Immature embryos contain significantly more soluble proteins than their derived tissue cultures (Table I). On immunoblots of soluble proteins from immature embryos, we observed a strong signal for NDPK (16 kD), a weak signal (58 kD) for Ga-proteins, and a faint signal (40 kD) for arrestin-like molecules. Similarly, immunoblots provided very weak signals for these three molecules from the embryo-derived membrane protein fraction.

Soluble Proteins as a Marker of Wheat Somatic Embryogenesis

Significant differences were noted in the content of total soluble proteins between immature embryos, embryogenic cultures, and nonembryogenic calli (Table I). The embryogenic cultures were discriminated from nonembryogenic cultures by their higher content of soluble proteins, whereas there was no difference in the SDS-PAGE profiles of the extracts (not shown). Experimental results suggest that the total soluble protein content is a simple, reliable marker of the embryogenic potential of wheat somatic tissue cultures.

The membrane protein content also reveals significant differences between embryogenic and nonembryogenic cultures. The embryogenic cultures possess two to three times more membrane proteins per gram fresh weight than the nonembryogenic cultures. The highest membrane protein content was observed for the immature embryos (two times more than the embryogenic cultures), again, on a fresh weight basis.

Table 1. Total soluble and insoluble protein content in wheat somatic tissues cultured in vitro Means ? 3 SE (99% confidence interval) were calculated from 6 to 10 independent experiments (only 3 for the dry weight).

Cenotype CS lmmature Embryos CS Embryogenic CS DT3DL CS DT3DS CS Nonembryogenic

Somatic embryogenesis nd" Yes Yes No No Soluble protein contentb 11.8 i 2.1 7.9 2 1.6 8.0 2 1.4 5.5 5 1.5 4.0 F 1.6

80.4 2 10' 49.2 ? 8.7' Membrane protein contentb 1.3 -t 0.4 0.6 i 0.2 nd nd 0.2 ? 0.1 a nd, Not determined. In mg of protein per g fresh weight. In mg of protein per g dry weight.



1804 Nato et al. Plant Physiol. Vol. 113, 1997

C-Protein Immunoreactivity

The antibody to the Ga-protein reveals two intenselystained bands in soluble extracts with apparent molecularmasses of 58 and 32 kD (Fig. 2A). The 58-kD band waspresent in the immature embryos and in all of the tissuecultures. The 32-kD band was detected in all of the cul-tures, including the proliferating nonembryogenic DT3DScallus (not shown), but was not observed in the immatureembryo extract. This significant tissue culture effect wasalso observed in the membrane protein fractions (Fig. 2B),where several bands were stained, including a major 58-kDband, in both embryogenic and nonembryogenic tissuecultures; there were weaker signals in the immatureembryos.

Arrestin Immunoreactivity

The mixture of arrestin mAbs reacted with a 40-kD sol-uble protein in all of the cultures tested (Fig. 3A). ThemAbs S2D2 (N-terminal epitope, amino acids 40-50) andM3A9 (intermediate, 263-282) also reacted with this 40-kDprotein (data not shown). There was no difference betweenthe CS embryogenic cultures, the CS nonembryogenic cal-lus, and the ditelosomics. On the contrary, the mAbs re-vealed an additional positive signal (29 kD) only in thesoluble proteins from the embryogenic cultures tested (CSand DT3DL).

The membrane fraction (Fig. 3B) contained the 40-kDprotein in both embryogenic and nonembryogenic cul-tures, with a weak signal for the immature embryos. Astrong signal at the 29-kD level was detected only in theembryogenic cultures. The 29-kD band appeared in solubleand membrane fractions after 3 weeks of in vitro culture(data not shown).

The mAb S1A3 cross-reacted with the 29-kD solubleprotein from embryogenic cultures (Fig. 4A, lane 2). On thecontrary, the polyclonal anti-LHCPII antibody did not reactwith any soluble protein from embryogenic cultures (Fig. 4,lanes 4). In addition, the mAb S1A3 also cross-reacted with

B

kDa 1

97 -•i« i«

50

35 -

29 -

22 -

Figure 2. Western blot analysis of soluble proteins (A) and mem-brane proteins (B) from wheat tissues using polyclonal antibodiesraised against a synthetic peptide corresponding to a highly con-served epitope of the mammalian a subunit of heterotrimericG-protein. Lanes 1, CS immature embryos; lanes 2, CS nonembryo-genic callus; and lanes 3, CS embryogenic culture.

k D a 1 2 3 4 5 6

22 -

Figure 3. Western blot analysis of soluble proteins (A) and mem-brane proteins (B) from wheat tissues using a panel of mAbs raisedagainst bovine arrestin epitopes. Lanes 1, CS immature embryos;lanes 2, CS nonembryogenic callus; and lanes 3, CS embryogenicculture. A, Lane 4, CS DT3DS nonembryogenic callus; lane 5, CSDT3DL embryogenic culture; and lane 6, purified bovine arrestin. B,Lane 4, purified bovine arrestin.

membrane proteins from embryogenic cultures (very largeband of 28-32 kD), whereas a thin signal was observedusing the polyclonal anti-LHCPII antibody against thesemembrane proteins (Fig. 4B).

Aneuploid Analysis

To ascertain the chromosomal location of the gene(s)encoding the arrestin-immunoreactive proteins, we used 36DT and 6 NT CS lines. This collection covers nearly all ofthe wheat chromosome arms, with the exception of 4ALand SAL. Among these lines, only the DT3DS is unable toproduce somatic embryos during in vitro culture. All of theother aneuploids have embryogenic capacities, even after along-term culture (Henry et al., 1994).

All of the aneuploid tissue cultures tested during theseexperiments, including DT3DS, revealed a positive signalwith a 40-kD protein. In contrast, the DT3DS nonembryo-genic callus was the only aneuploid that did not exhibitimmunoreactivity corresponding to the 29-kD arrestin-likepolypeptide (Fig. 3A, lane 4). On the contrary, all of the

BkDa 1

97 -50-

35 -29-

22 -

kDa 1 2

97-50-

35-

29-

22-

Figure 4. Western blot analysis of soluble proteins (A) and mem-brane proteins (B) from wheat tissues using both the mAb S1 A3 raisedagainst bovine arrestin epitope 371 to 380 (lanes 1 and 2) and thepolyclonal antibody raised against CWamvc/omonas-purified LHCPII(lanes 3 and 4). Lanes 1 and 3, CS nonembryogenic callus; lanes 2and 4, CS embryogenic cultures.



Immunological Detection of Potential Signaling Elements 805

other 35 DT and 6 NT lines tested, which have embryogenicpotential, showed a positive cross-reaction signal at 29 kD(data not shown).

NDPK Immunoreactivity

On immunoblots, all of the tested materials (immatureembryos, CS embryogenic, CS DT3DL, CS nonembryo-genic, and CS DT3DS) contained a 16-kD protein detectedby the 143-2 mAb in their soluble protein extract (Fig. 5).This suggests that wheat possesses at least one majorNDPK form. Moreover, another polypeptide band was de-tected at a higher molecular mass (32 kD) in tissue cultures(Fig. 5A).

In the insoluble protein fraction a 16-kD band was clearlydetected in the nonembryogenic and embryogenic cultures.This band was very weak in the immature embryo insolu-ble fraction (Fig. 5B).

DISCUSSION

Under in vitro conditions auxin is necessary for theinduction of embryogenesis from wheat immature embryocells. The hormone binds to a signal receptor coupled toG-protein (Guern et al., 1990; Millner, 1995) and severalkinases participate in integrating the signal (Lefkowitz,1993; Pelech et al., 1993). As a consequence of this trans-duction pathway, the development of the somatic cells isdisrupted and some of them acquire embryogenic poten-tial. The study of plant signal-transduction pathways re-lated to embryogenesis is of major interest for understand-ing development.

The sequence of the Ga-protein oligopeptide that weused to generate an immune serum was compared withthose from known plant heterotrimeric Ga-protein sub-units. The deduced amino acid sequences of the productsof GPA1 (Ma et al., 1990), TGA1 (Ma et al., 1991), and RGAl(Ishikawa et al., 1995; Seo et al., 1995) reveal a high degreeof homology to a subunits of mammalian heterotrimericG-proteins in the region that binds to GTP (Ishikawa et al.,1995) (Fig. 1). The consensus region GxxxxGK(S/T) for

BkOa . 197 .50 .

35 -29 -

22 - 22-

Figure 5. Western blot analysis of soluble proteins (A) and mem-brane proteins (B) from wheat tissues using the 143-2 mAb raisedagainst NDPK. Lanes 1, CS immature zygotic embryos; lanes 2, CSnonembryogenic callus; and lanes 3, CS embryogenic culture. A,Lane 4, Dictyostelium pure NDPK.

GTP binding, found in the a subunit of heterotrimericG-proteins (Ma, 1994), is also present in highly conserved,Ran-soluble, small G-proteins from human cells, yeast, andplants (Ach and Gruissem, 1994; Merkle et al., 1994; Saal-bach and Christov, 1994), which have functions both inregulating the mitotic cycle and in protein import into thenucleus (Merckle et al., 1994).

It has been shown that putative heterotrimericG-proteins are overexpressed during early embryogenesisin Xenopus (Paquereau and Audigier, 1995) and that a highlevel of the Ga subunit can be observed in the developingembryos of Arabidopsis (Weiss et al., 1993). The antiserumto the Ga subunit labeled a 58-kD protein in soluble andmembrane extracts from wheat immature embryos andsomatic tissue cultures and a 32-kD protein that waspresent in the soluble fraction of cultured tissues. Themolecular mass of the 58-kD protein is high compared withthose of animal Ga proteins, but such high molecularmasses of Ga subunits have been mentioned in plants: 112kD (Ma, 1994) and 68 kD (Dombrovsky and Raikel, 1995).

The additional low-molecular-mass (32 kD) form ap-peared in all of the tissue cultures and can be interpreted asbeing induced by the in vitro conditions (auxin presence).The appearance of this new form suggests a presumedfunction similar to that of the family of ras-relatedG-proteins, which are known to control cell cycle, cellgrowth, differentiation, and the organization of the cy-toskeleton (Drayer and van Haastert, 1994).

Western blot analysis of all of the wheat material testedusing N-terminal and central anti-arrestin mAbs on solubleand membrane protein fractions revealed the presence ofan immunoreactive protein with a molecular mass of 40kD. This is similar to the molecular mass of 39 kD for D.melanogaster arrestin 1 (Matsumoto and Yamada, 1991).Moreover, it has been demonstrated that human and bo-vine arrestins as well as j3-arrestins possess truncated andextended forms generated by alternative mRNA splicing(Palczewski et al., 1994).

Western blots also clearly showed an additional form(29-kD protein), expressed both in soluble and membraneprotein fractions and associated with cultured wheat tis-sue, that has embryogenic capacity. Our experiments using42 aneuploid lines (DT and NT) suggested that the 3DLchromosome arm, which is essential for somatic embryo-genesis (Henry et al., 1994), also possesses a gene thatencodes the soluble 29-kD protein or controls its expres-sion. The detection of this 29-kD protein could allow dis-crimination between embryogenic and nonembryogenictissues. This explanation is attractive, but an alternativecould be a general, indirect consequence of the DT3DS cellsbeing nonembryogenic.

Because the anti-LHCPII antibody did not recognize anyprotein in the soluble extracts, we speculate that the 29-kDlight-dependent protein detected by the anti-arrestin anti-body in these soluble preparations could belong to thearrestin family as a component of a photomorphogeneticsignal-transduction pathway involving the activity ofG-proteins (Bossen et al., 1990; Mac Nellis and Deng, 1995).The very large band (28-32 kD) detected in the membrane www.plantphysiol.orgon July 29, 2018 - Published by Downloaded from

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806 Nato et al. Plant Physiol. Vol. 11 3 , 1997

protein fraction from embryogenic cultures by the anti- arrestin antibody could be composed of severa1 proteins. Indeed, a 29-kD band is revealed by the anti-LHCPII antibody, suggesting that at least one cross-reacting protein belongs to the light-harvesting complex LHCPII (M. Mir- shahi, unpublished data).

Recent studies have linked NDPK with G-proteins through phosphorylation of enzyme-bound GDP (Kimura and Shimada, 1990; Finan et al., 1994). The low-molecular- mass 16-kD putative NDPK form detected in the soluble and membrane protein fractions from wheat tissues could be related to the 16-kD form previously described (Nomura et al., 1992; Finan et al., 1994) and to the 18-kD NDPK from sugarcane cells (Moisyadi et al., 1994). The wheat immature embryos revealed a reduced amount of the 16-kD membrane form compared with in vitro-cultured cells. The activity of a receptor-stimulated Dictyostelium NDPK contrib- utes to the mediation of hormonal action (Bominaar et al., 1993), so the increase observed for the 16-kD form in the wheat tissue cultures could be related to an auxin effect. An additional band (32 kD) is mostly expressed in soluble protein extracts from CS embryogenic cultures, suggesting a putative function in cell differentiation regulation during tissue culture.

Interest in the wheat G-protein, arrestin-like molecules, and NDPK has increased as a result of their presumed involvement in cell proliferation and differentiation. We are currently studying spatial and temporal patterns of these putative signaling elements in the presence or ab- sence of light and hormones. Advances with our plant system will also require, first, the development of biochemical assays, such as enzyme assays for NDPK and binding assays for GTP-binding proteins, and, second, the molecular cloning of cDNAs and sequence determination of the observed proteins. Molecular and biochemical studies of embryogenesis are central points in plant developmental biology.

ACKNOWLEDCMENTS

The authors would like to thank Odile Jeannequin, François Traincard, and Michel Veron (Hybridolab and Unité de Biochimie Cellulaire, Institut Pasteur, Paris) for providing the 143-2 NDPK mAb and Guy Dubertret and Antoine Trémolières (Unité de Re- cherche Associée Centre National de la Recherche Scientifique, Orsay, France) for providing the Chlamydomonas antiserum against LHCPII. The authors are grateful to Prof. Martin Kreis for helpful suggestions and critica1 reading of the manuscript.

Received July 15, 1996; accepted November 25, 1996. Copyright Clearance Center: 0032-0889/97/113/0801/07.

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