Arginine Decarboxylase 1s Localized in Chloroplasts’ · Arginine Decarboxylase 1s Localized in Chloroplasts’ ... plasm and the nucleus (Gilad and Gilad, ... The synthesis of Spd

Plant Physiol. (1 995) 109: 771-776

Arginine Decarboxylase 1s Localized in Chloroplasts’

Antonio Borrell, Francisco A. Culiaííez-Macia, Teresa Altabella, Robert T. Besford, Dante Flores, and Antonio F. Tiburcio*

Laboratori de Fisiologia Vegetal, Facultat de Farmicia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain (A.B., T.A., D.F., A.F.T.); Departament de Genètica Molecular, Centre d’ Investigació i

Desenvolupament, Consejo Superior de lnvestigaciones Científicas, Jorge Girona 1 8-26, 08034 Barcelona, Spain (F.A.C.-M.); and Horticulture Research International, Littlehampton, West Sussex, BN 17 6LP,

United Kingdom (R.T.B.)

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC.

Polyamines are polycationic cellular molecules that play an essential role in cell growth and differentiation (Pegg, 1986; Auvinen et al., 1992). Put, the precursor of polyamines, is formed in animals only by decarboxylation of Orn, via ODC (EC 4.1.1.17) (Heby and Persson, 1990). In contrast, in plants and bacteria there is an alternative pathway by which Put is produced from the decarboxylation of Arg by ADC (EC 4.1.1.19) (Tabor and Tabor, 1985; Tiburcio et al., 1990).

In plants, polyamines, formed by either ADC or ODC, or both, are important modulators of biological responses such as cell division, reactions to stress, and development (Galston and Kaur-Sawhney, 1990; Galston and Tiburcio, 1991). However, the precise mechanism of their involve-

* This work was supported by Comisión Interministerial de Ciencia y Tecnología BI093-130, European Union-Concerted Ac- tion Programme AIR1-CT92-569 to A.F.T., by the British-Spanish Joint Research Program “Acciones Integradas” HB-079, HB-119A (R.T.B. and A.F.T.) and by the Biotechnology and Biological Sci- ences Research Council (R.T.B.).

* Corresponding author; e-mail afernan8farmacia.ub.es; fax 34- 3-402-1886.

ment in such diverse phenomena is not clear. Lack of knowledge of the exact cellular and subcellular localization of polyamine biosynthetic enzymes has been one of the main obstacles in our understanding of the biological role of the ODC/ ADC/ polyamine system. In animals, cytochemical, autoradiographic, and immunocytochemical evidence suggests that ODC is localized in both the cytoplasm and the nucleus (Gilad and Gilad, 1981; Pegg et al., 1982; Persson et al., 1983; Dodds et al., 1990). To our knowledge, in plants, there is only one study on the autoradiographic localization of ODC in the nucleus and cytoplasm (Slocum, 1991). Little is known about the localization of ADC protein, despite its wide occurrence in plants (Tibur- cio et al., 1990). The synthesis of Spd and Spm is carried out by addition of an aminopropyl moiety to one or both primary amino groups of Put by Spd synthase (EC 2.5.1.16) and Spm synthase (EC 2.5.1.22), respectively. Decarboxy- lated SAM, which acts as the aminopropyl donor, is derived from SAM via the action of SAM decarboxylase (EC 4.1.1.50) (Tiburcio et al., 1990). As for ADC, little is known about the localization of these enzymes (Slocum, 1991).

It is now well documented that a variety of physiological stimuli and stress reactions affect the activity of polyamine biosynthetic enzymes in higher plants (Flores and Galston, 1982; Tiburcio et al., 1990; Slocum and Flores, 1991). How- ever, little is known about the metabolic and molecular regulation of biosynthesis of these enzymes in plants (Slo- cum and Flores, 1991). For example, ADC activity in tobacco cells was fully repressed by exogenous Spd or Spm, without affecting ODC activity; however, the precise mechanism of ADC inhibition by polyamines is unknown (Hiatt et al., 1986). On the other hand, the factors determining the flux rates through the ADC versus ODC pathways in plants are still terra incognita (Slocum and Flores, 1991). In bacteria, flux through the ADC and ODC pathways ap- pears to be determined by Orn availability, since intracellular Arg levels are always high (Tabor and Tabor, 1985). Under normal conditions, where Orn levels are not limit- ing, Put is synthesized primarily by ODC. If the Orn con- centration drops, Arg is then utilized in Put synthesis via

Abbreviations: ADC, arginine decarboxylase; DFMA, DL-a-di- fluoromethylarginine; DFMO, DL-a-difluoromethylornithine; ODC, ornithine decarboxylase; Put, putrescine; SAM, S-adenosyl- methionine; Spd, spermidine; Spm, spermine.

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772 Borrell et al.

the ADC pathway. In the plant cell, flux through these pathways may be determined by different factors, includ- ing those derived from compartmentalization of intermediates or enzymes of Put synthesis (Slocum and Flores, 1991).

with the aim of understanding the molecular mechanisms regulating ADC gene expression by polyamines, we generated polyclonal antibodies to oat (Avena sativa L.) ADC to investigate changes in ADC protein levels in osmotically stressed oat leaves (Tiburcio et al., 1994a). The results suggested that Spm affects the synthesis of ADC by inhibiting the posttranslational proteolytic processing of the inactive ADC precursor with a consequent decrease in the active, processed form of ADC (Tiburcio et al., 1994a). Here we describe the use of our anti-ADC antibodies to study the spatial pattern distribution and subcellular compartmentalization of ADC protein in different oat tissues. We report evidence that ADC is localized in the chloroplast compartment. This finding opens new insights into the catabolism of Arg in photosynthetic tissues.

MATERIALS A N D METHODS

Plant Material and Osmotic Treatment

Avena sativa L. cv Victory (Svalof International, Svalov, Sweden) plants were grown as described elsewhere (Besford et al., 1993). Plants whose leaves were used for isolating chloroplasts were kept in the dark for 12 to 18 h to allow depletion of starch and were sampled at the end of the dark period.

For osmotic treatment, peeled oat leaves were dark-incubated with a phosphate buffer containing 0.6 M sorbitol for 4, 24, or 48 h (Besford et al., 1993).

Subcellular Fractionation

Leaves (15 g) were cut into small pieces with scissors and homogenized in 30 mL of a buffer containing 0.35 M SUC, 25 mM Hepes, 2 mM EDTA, pH 7.6. Homogenates were fil- tered through two layers of Miracloth (Calbiochem) and centrifuged at lOOg for 2 min at 4°C to separate nuclei and cell-wall materials (Choe and Thimann, 1975). The residue was discarded, and the supernatant was centrifuged at 40008 for 1 min at 4°C. Both supernatant and enriched chloroplast pellet fractions were analyzed by immunoblot.

In another experiment, we purified intact chloroplasts from an enriched chloroplast pellet obtained as described above. This pellet was resuspended in 10 mL of 0.33 M

sorbitol and 50 mM Hepes-KOH, pH 7.5, and 5 mL were layered over each of two 30-mL Percoll gradients, prepared as described by Robinson and Barnett (1988). The Percoll- purified chloroplasts were lysed with 12 mL of 10 mM Tricine-KOH, pH 7.8, 4 mM MgCI,, and 1 mM PMSF. After addition of 1.8 mL of 80% (w/v) SUC, the mixture was layered onto a discontinuous SUC gradient of 0.98 and 0.6 M

SUC and centrifuged at 90,OOOg for 2 h. Under these conditions, the thylakoid and soluble stromal fractions appeared, respectively, at the bottom and at the top of the gradient. An aliquot of each fraction was subjected to SDS-PAGE and immunoblotting.

Plant Physiol. Vol. 109, 1995

Ceneration of Antibodies

An amino acid sequence (Gly-Pro-Ser-Leu-Val-Arg- Val-Val-Gly-Thr-Gly-Asn-Gly-Gly-Ala-Phe-Asn-Val-Glu -Ala) near the C terminus deduced from the nucleotide sequence of the oat ADC gene (Bell and Malmberg, 1990) was se- lected. This polypeptide was synthesized by solid-phase peptide synthesis, coupled to purified-protein-derivative of tuberculin carrier protein, and injected into rabbits to produce the corresponding polyclonal antibodies. The method is fully described elsewhere (Besford et al., 1990).

To test the specificity of the antiserum, anti-ADC antibodies were covalently linked to protein A-Sepharose (Pharmacia) as described by Schneider et al. (1982). ,4fter immunoprecipitation with either preimmune serum or with anti-ADC antibodies, ADC activity was determined as described below.

lmmunoblot Analysis

Proteins were extracted from plant material with a buffer containing 0.6% P-mercaptoethanol and 1% SDS in 20 mM Tris buffer, pH 8.6, boiled for 5 min, and centrifuged at 15,OOOg for 5 min. The supernatants were subjected to SDS-PAGE and immunoblotting. SDS-PAGE (15% acryl- amide) was performed according to Laemmli (1970). Im- munoblot analyses were carried out as described by Besford (1990).

Analysis of ADC and O D C Activities

ADC and ODC activities were determined as described elsewhere (Tiburcio et al., 1986). No significant release of CO, was observed in enzyme extracts preincubated with DFMA or DFMO. Therefore, possible artifacts (Birecka and Birecki, 1993) resulting from nonspecific decarboxylation of enzyme extracts were eliminated.

lmmunolocalization of ADC Protein

For immunolocalization, the immune serum obtained against the ADC protein was used. An all-purpose fixative (80% ethanol, 3.5% formaldehyde, 5% acetic acid) was used for paraffin embedding. Sections from paraffin-embedded material were blocked with 3% goat serum in PBS (10 mM phosphate, 150 mM NaC1, pH 7.4) for 30 min at 22°C' and incubated with anti-ADC immune serum (diluted 1:500) or preimmune serum (diluted 1:500). Immunoreactivity was visualized by the avidin-biotin complex (Vectastain Elite ABC Kit, Vector, Burlingame, CA) using diaminobenzidine as substrate for peroxidase.

RESULTS

Organ-Specific Accumulation of ADC

We generated site-directed polyclonal antibodies to oat ADC (Tiburcio et al., 1994a). The primary structure 0.f oat ADC (Bell and Malmberg, 1990) was used for the selection of an epitope by computer calculations using the Genetics Computer Group (Madison, WI) package. The choice of this epitope was based on predictions of antigenicity and

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Localization of Arg Decarboxylase 773

the fact that this region does not contain substrate bindingsites (Tiburcio et al., 1994b).

Immunoprecipitation of the ADC protein present in oatleaf extracts resulted in a parallel decrease of the ADCactivity of those extracts (Fig. 1). These results reflect thespecificity of the antiserum used in this study.

Proteins from leaves and root of oat seedlings wereextracted, separated by SDS-PAGE, and analyzed by im-munoblotting using our anti-ADC antibodies. Figure 2shows that there was cross-reactivity, which is apparentlyleaf specific. A single, 24-kD band was recognized inleaves, whereas in roots no band was detected (Fig. 2).These results agree with the high levels of ADC activityfound in leaves (5 nmol CO2 h ' mg"1 protein) comparedto the negligible levels of ADC activity found in roots.Previous work has shown that oat ADC is synthesized as apreprotein of 66 kD, which is cleaved to produce a frag-ment of 42 kD (containing the original amino terminus)and a 24-kD polypeptide (containing the original carboxylterminus), these two processed polypeptides being heldtogether by disulfide bonds (Malmberg et al., 1992; Malm-berg and Cellino, 1994). Thus, the 24-kD band detected inour protein gel blots, run with /3-mercaptoethanol, repre-sents the processed form of the ADC enzyme.

The spatial distribution of ADC protein in different or-gans of oat seedlings was confirmed by immunocytochem-istry. Bright-field photographs of paraffin sections from9-d-old seedlings incubated with anti-ADC antibodiesshow that ADC protein was detected in oat leaves andstems (Fig. 3, A and C) but not in roots (Fig. 3D). The ADCprotein appeared evenly distributed in the chloroplasts ofleaves (Fig. 3A), whereas in stems ADC was located pri-marily in perivascular chloroplasts (Fig. 3C). The resultsobtained with oat stem led us to test whether ADC could bedifferentially located in leaves of C4 plants containing dif-ferent functional chloroplasts (Salisbury and Ross, 1991).We took advantage of the cross-reaction of the oat ADCantibody with corn (a C4 plant), in which it recognized asingle band in immunoblots, with a molecular mass similarto that of oat ADC. In corn, as in oat stem, the oat ADC

75-

25-

"-0-.,.

5 10 15 20

Anti-ADC antiserum !/(ll

25

Figure 1. Immunodepletion of ADC. ADC activity of oat leaf extractsremaining in the supernatant after immunoprecipitation with eitherpreimmune serum (D) or anti-ADC antibodies (0).

24 »-

Figure 2. Immunoblot analysis of protein extracts from oat leavesand roots using ADC antibodies. Lane 1, Leaves from 9-d-old seed-lings; lane 2, roots from 9-d-old seedlings; lane 3, molecular massmarkers: 106, 80, 49, 32, 27, and 18 kD. Each lane was loaded with30 ,u,g of protein.

antibodies reacted strongly with perivascular chloroplasts,although no reaction was seen with the mesophyll chloro-plasts (data not shown).

ADC Protein Localized in Chloroplasts

To confirm the intracellular location of ADC protein,chloroplasts were purified from leaves of 9-d-old oat seed-lings by differential centrifugation, and supernatant andchloroplast-enriched pellet fractions were analyzed withanti-ADC antibodies after PAGE and immunoblotting (Fig.4). A unique band of 24 kD was detected in the pelletcontaining chloroplasts, whereas no band was detected inthe supernatant (Fig. 4, lanes 2 and 3). In different experi-ments we determined the intrachloroplastic location ofADC protein. We first obtained the enriched chloroplastpellet, which was further resuspended in a Hepes-sorbitolbuffer and fractionated on a discontinuous Percoll gradi-ent. The purified chloroplasts were then lysed and fraction-ated on a Sue gradient. The resulting soluble stromal andthylakoid fractions were analyzed by immunoblotting (Fig.4). The 24-kD band was detected primarily in the thylakoidfraction, whereas only a faint band was detected in thestromal fraction (Fig. 4, lanes 4 and 5).

These results were further confirmed by immunocyto-chemistry at the electron-microscope level. Gold particle-labeled ADC antigen appeared mostly over the thylakoidmembranes, whereas some label was observed on the restof the chloroplast (data not shown). How ADC is associ-ated with the thylakoids is not known, but studies on theADC secondary protein structure, using the Genetics Com-puter Group package, have shown some putative am-phiphilic-a-helix-forming regions with a high hydrophobicmoment, which might be involved in membrane interac-tions. However, no clear transmembrane domains havebeen found. www.plantphysiol.orgon June 7, 2018 - Published by Downloaded from

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774 Borrell et al. Plant Physiol. Vol. 109, 1995

B

r X

#

Figure 3. Immunolocalization of ADC protein. Paraffin-embedded sections of organs from 9-d-old oat seedlings wereincubated with anti-ADC (A, C, and D) or preimmune antiserum and an avidin-biotin-peroxidase detection system. Brownstaining indicates the presence of peroxidase activity. A and B, Leaf sections; C, stem transverse section (magnification X570); D, root section (magnification x 32).

Response of ADC Enzyme to Osmotic Stress

Previous work has shown that osmotic treatment of oatleaves in the dark increases the levels of ADC activity andleads to senescence (Tiburcio et al., 1994b). Hence, in thisstudy changes in soluble ADC protein levels of oat leavessubjected to osmotic treatment were analyzed. Osmoticstress increased the levels of the 24-kD polypeptide incomparison with the 0-h control (Fig. 5). No interferingbands were detected after immunoblotting with preim-mune serum (data not shown). These changes in ADCprotein levels are well correlated with changes in ADCactivity and Put levels (Tiburcio et al., 1994b).

DISCUSSION

Although ADC is apparently ubiquitous in plants, itssubcellular location has not been definitely determined.Previous work attempting the subcellular localization ofADC involved the measurement of enzyme activity (Tor-rigiani et al., 1986; Walker et al., 1987). However, these

studies have been questioned because of artifacts resultingfrom the use of 14C trapping (Birecka and Birecki, 1993;Smith, 1993).

Here we show that ADC distribution is organ specific,apparently localized to the thylakoid membranes of thechloroplast. This has been demonstrated by cell fraction-ation and immunocytochemistry at the electron-micro-scope level. This indicates that a plant-specific polyaminebiosynthetic pathway is located in the chloroplast, andraises the intriguing question of whether Put is synthesizedin root tissue de novo or transported there.

Figure 6 shows a proposed model for the Arg pathwayand Put biosynthesis in active photosynthetic tissues. Theprimary enzymes of the Arg pathway (from glutamate tocitrulline) are chloroplastic, whereas the terminal step fromcitrulline to Arg is carried out in the cytoplasm (reviewedby Bryan, 1990). Our study indicates that ADC is chloro-plastic; therefore, transport of Arg from the cytoplasm tothe chloroplast and further decarboxylation of this aminoacid by ADC provides the only source of newly synthe- www.plantphysiol.orgon June 7, 2018 - Published by Downloaded from



Localization of Arg Decarboxylase 775

1 2

-24

Figure 4. Immunoblot analysis of ADC protein in protein extractsfrom different subcellular fractions. Lane 1, molecular mass markers:106, 80, 49, 32, 27, and 18 kD; lanes 2 and 3, supernatant andchloroplast-enriched pellet fractions; lanes 4 and 5, thylakoid andstromal fractions. Each lane was loaded with 30 jxg of protein.

sized Put (and polyamines) within this organelle (Fig. 6).This hypothesis is further supported by the fact that neg-ligible levels of ODC activity were detected in oat leaves.

In contrast, in a nonchloroplastic tissue (such as oatroots) the levels of ODC activity are high (4 nmol CO2 h"1

mg"1 protein) in relation to the negligible levels found inleaves. Therefore, in the absence of ADC, the enzyme ODCmay be the key enzyme of Put biosynthesis in roots.

In Neurospora (a nonphotosynthetic organism), most ofthe primary enzymes of the Arg pathway are localized tothe mitochondria, but the terminal step from citrulline toArg is carried out in the cytoplasm (Schubert and Boland,1990). In the absence of ADC, Arg can be catabolized by theenzyme arginase (EC 3.5.3.1) to urea and Orn (Lehninger,1980), and this amino acid may be further metabolized toPut and polyamines. In fact, Arg in mammals is consideredan essential amino acid, since it is rapidly catabolized byarginase and is usually not available for protein synthesis(Lehninger, 1980). Therefore, Orn may be the only sub-strate available for the synthesis of Put in mammals. Thislast model may also operate in roots and other nonphoto-synthetic tissues.

24-

TFigure 5. Immunoblot analysis of ADC protein in osmoticallystressed oat leaves. Protein extracts from osmotically treated oatleaves incubated with immune serum. Treatment with 0.6 M sorbitol:0 time (lane 1); 4 h (lane 2); 24 h (lane 3); and 48 h (lane 4).Molecular mass markers: 106, 80, 49, 32, 27, and 18 kD (lane 5).Each lane was loaded with 50 jig of protein.

GLUTAMATE

'It"AG

AGP

• tAGSA

AORN6 [ ———

2Chloroplast

Polyamines

f* 11 ' 10

ORN--------------»- PUT-« —— -« ——— •« —— ARGININE

err

" 1 . . 1 "CIT ———— -7 ———————— ** AS ————— - —————— f- ARGININE

ASPCytosol

Figure 6. Proposed scheme for compartmentalization of Argpathway and Put biosynthesis in actively photosynthetic tissues.Intermediates: AG, N-acetylglutamate; AGP, N-acetylglutamate5-phosphate; AGSA, N-acetylglutamate 5-semialdehyde; AORN,N-acetylornithine; ORN, Orn; CIT, citrulline; ASP, aspartate; AS,argininosuccinate; PUT, Put. Enzymes: 1, acetyl-CoA:glutamate N-acetyltransferase (EC 2.3.1.1); 2, acetylornithine:glutamate N-acetyl-transferase (EC 2.3.1.35); 3, N-acetylglutamate kinase (EC 2.7.2.8);4. N-acetylglutamate semialdehyde oxidoreductase (EC 1.2.1.38);5. N-acetylornithine aminotransferase (EC 2.6.1.11); 6, acetyl-ornithine deacetylase (EC 3.5.1.16); 7, ornithine carbamoyltrans-ferase (EC 2.1.3.3); 8, argininosuccinate synthase (EC 6.3.4.5); 9,argininosuccinate lyase (EC 4.3.2.1); 10, Arg decarboxylase; 11,Orn decarboxylase.

In the context of the endosymbiote-driven metabolic evo-lution in plants (Weeden, 1981), the finding that ADC islocalized in the chloroplast may have a major evolutionarysignificance, since it explains (a) why plants can use twoalternative pathways for the synthesis of the same com-pounds and (b) why animals, irrespective of precursoravailability, do not possess ADC.

Chloroplasts have the capacity to encode and synthesizeonly some of their proteins, and the remainder are proba-bly all nuclear encoded (Dyer, 1984). These nuclear-en-coded proteins are synthesized in the cytoplasm, generallyas larger precursors containing a transit peptide, and enterchloroplasts via a posttranslational process (Keegstra et al.,1995). Since oat ADC mRNA is poly(A)+ (Bell and Malm-berg, 1990), ADC protein may be nuclear encoded, raisingthe question of entrance to the plastid, which is currentlybeing investigated.

ACKNOWLEDGMENTS

Our thanks to Robin Rycroft for help in preparing the manu-script and Rick Walden, Maarten Chrispeels, Montse Pages, andCarles Masgrau for their comments.

Received May 9, 1995; accepted August 7, 1995.Copyright Clearance Center: 0032-0889/95/109/0771/06. www.plantphysiol.orgon June 7, 2018 - Published by Downloaded from



776 Borrell et al. Plant Physiol. Vol. 109, 1995

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Arginine Decarboxylase 1s Localized in Chloroplasts’ · Arginine Decarboxylase 1s Localized in Chloroplasts’ ... plasm and the nucleus (Gilad and Gilad, ... The synthesis of Spd