Phymchtminry. Vol. 35. No. 2, pp. 281.286, 1534 Copyright Q 1994 Ekna sdcwc Ltd

Printed m Chat Britain. All ri&ts mewed cm-942.2194 56.00+0.00

CELL WALL PROTEINS IN SEEDLING COTYLEDONS OF PROSOPIS CHILENSIS

Jose GREGORIO RODRIGUEZ* and LILIANA CARDEMIL?

Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile

(Received in revised form 27 May 1993)

Key Word Index-Prosopis chilensis; Leguminosae; algarrobo, extensin; proline-rich proteins; hydroxy proline-rich proteins; polyclonal antibodies; cotyledons; soybean seed coats.

AIrstract-Four cell wall proteins of cotyledons of Prosopis chilensis seedlings were characterized by PAGE and Western analyses using a polyclonal antibody, generated against soybean seed coat extensin. These proteins had M,s of 180000, 126000, 107000 and 63000, as determined by SDS-PAGE. The proteins exhibited a fluorescent positive reaction with dansylhydrazine suggesting that they are glycoproteins; they did not show peroxidase activity. The cell wall proteins were also characterized by their amino acid composition and by their amino-terminal sequence. These analyses revealed that there are two groups of related cell wall proteins in the cotyledons. The first group comprises the proteins of Mp 180 000, 126 OCO, 107 000 which are rich in glutamic acid/ glutamine and aspartic acid/asparagine and they have almost identical NH,-terminal sequences. The second group comprises the M, 63 000 protein which is rich in proline, glycine, valine and tyrosine, with an NH,-terminal sequence which was very similar to that of soybean proline-rich proteins.

INTRODUCTION

Prosopis chilensis, also known as algarrobo, is a native tree from the arid and semiarid regions of central and northern Chile. The tree has economic importance due to

the high quality of its timber and as a source of proteins for livestock. One of the physiological events important in the study of propagation and plant survival is the expression of cell wall proteins since they are considered to play a role in protection against plant infection [1] and in lignification and tissue repair after wounding [2].

From the time that Lamport discovered [3], isolated and sequenced the first peptides of the extensins [4] which are structural proteins present in the cell wall of most higher plants, five classes of cell wall proteins have been described: hydroxyproline-rich glycoproteins (HRGPs) or extensins [S, 63, glycine rich-proteins [7], proline-rich proteins [8, 93, Solanaceae lectins [lo] and arabinogalactan proteins [ 111.

Prosopis chilensis is a tree described as being resistant to heat stress, water drought, salinity [12] and to injury stress (field observations). For these reasons, we wanted to evaluate the expression of cell wall proteins in cotyle- dons of P. chilensis seedlings which are predated by small mammals, birds and insects and, therefore, injured. In this work, we report a partial biochemical characterization of four cell wall proteins present in cotyledons from seedlings of this leguminous tree.

*Present address: IDEA, Unidad de Biotecnologia, Apartado 17606, Parque Central, Caracas, Venezuela.

tAuthor to whom correspondence should be addressed

RESULTS

Cell wall protein pattern

The amount of hydroxyproline in the proteins ex- tracted from cotyledonary walls of P. chilensis 24 hr after germination was 20 pg g- ’ of the fresh weight, with a specific concentration of 0.1 pg pg- ’ of cell wall protein. SDS-PAGE analysis of the proteins extracted revealed the presence of 13 protein bands. However, only a few of these proteins penetrated and ran in their native forms when analysed by electrophoresis performed under ca- tionic neutral conditions (Fig. IA). Of these, four bands cross-reacted with polyclonal antibodies raised against extensin protein from soybean seed coats (Fig. 1B). These four proteins ran in the same order on both SDS-PAGE and cationic neutral gels. SDS-PAGE analysis allowed the determination of their M,s as being 180 000, 126 000, 107 000 and 63 OCO.

Characterization of cell wall proteins

The glycoprotein nature of the cell wall proteins was demonstrated by incubation of the gel after SDS-PAGE with periodic acid and dansylhydrazine. The four pro- teins gave fluorescent bands with these reagents.

Peroxidase activity

None of the four proteins analysed gave a positive reaction when the cationic neutral gels were stained for enzyme activity with H,O, and o-phenylenediamine. In

281

282 J. G. RODRIGUEZ and L. CARDEMIL

-ND 63

Fig. 1. Cationic neutral gel electrophoresis and Western blot

analyses of the native cell wall proteins present in the cotyledons

of P. chilensis. (A) Gel stained with silver nitrate. (B) Western

analysis of the proteins shown in A. The proteins were electro-

transferred to nitrocellulose membranes and incubated firstly

with a rabbit antibody raised against extensin from soybean seed

coats and then incubated with a second antibody, a goat anti-

IgG conjugated with alkaline phosphatase. Arrows indicate the

corresponding bands of proteins in A and B. The numbers at the right are the M, values of each protein.

the walls, however, there were other protein bands of M,

lower than 25 000 which showed peroxidase activity.

Amino acid composition oj proteins

There were two classes of proteins in P. chilensis

cotyledons (Table I). The first class comprised the pro- teins of M,s 180000, 126000 and 107000, which were relatively rich in glutamic acid/glutamine, aspartic acid/aspargine, and with less (in decreasing order) argi- nine, proline, leucine and glycine, with the results ex- pressed either as mol per cent or as the number of residues per molecule. The other class comprised the M, 63000 protein which was rich in glutamic acid/glutamine and followed (in decreasing order) by proline, lysine, valine, aspartic acid/aspargine and tyrosine. All four proteins contained cysteine, an amino acid not present in the extensins reported so far [S, 63.

The amino acid compositions of the four proteins were compared with two other proteins present in soybean (G/y&e max) cell walls Cl33 (Table I). The M, 63000 protein was similar to the soybean cell wall proteins SbPRPl and ENODZ in its lower content of serine,

isoleucine, leucine and arginine. The protein, however, was closer to the SbPRPl than to the ENODZ from its higher content of valine and tyrosine.

NH ,-terminal sequence of proteins

Analysis of the NH,-terminal sequences of the four proteins also suggested the presence of the same two classes of cell wall proteins (Table 2). The class formed by the proteins of M,s 180000, 126 000 and 107 Ooo had a very similar sequence. The sequences of the proteins of MS 126000 and 107000 were almost identical and differed in only seven amino acids with respect to the protein of M, 180000. The NH,-terminal sequences of these proteins were unique, with no homology to any of the proteins present so far known.

The M, 63 000 protein had an NH,-terminal sequence clearly different from the first group. It has proline and hydroxyproline, amino acids which were not present in the other proteins. A comparison of this protein sequence using the FASTA aligment analysis [I43 demonstrated high homology in its NH,-terminal sequence with those of proline-rich proteins present in other dicotyledonous plants (Table 3). Proline-rich proteins present in cell walls of soybean are the ones with higher homologies to the NH,-terminal sequence of the M, 63ooO protein. A distance matrix revealed that the soybean cell wall pro-

tein coded by the genomic clone PRP2 [IS] is the one with higher homology to the M, 63000 protein of P. chilensis

(Table 4).

DISCUSSION

The M,s of the cell wall proteins of P. chilensis cotyledons are in the range reported for other hydroxyproline-rich glycoproteins present in plant cell walls [16]. The four cell wall proteins cross-reacted with antibodies raised against extensin from soybean seed coats, suggesting that these proteins are structural cell wall proteins of the extensin-type. The presence of hydroxyproline in the hydrolysates of cell wall extracts corroborated the presence of extensin-like proteins in the cotyledonary walls.

Although peroxidases (EC I.1 1.1.7) are commonly present in the cell walls of many plants [17], it is unlikely that the four cell wall proteins of P. chilensis belong to this class of enzyme because they did not show any peroxidase activity, although peroxidase activity was found in other proteins of the cell walls. These peroxidases could be responsible for insolubilization of extensin-like proteins in the walls as has been reported [6, 17, IS]. Indeed, there are hydroxyproline-containing proteins in P. chilensis cell walls which cannot be extracted with 0.2 M CaCI,.

According to Kieliszewski and Lamport [IS], polyclo- nal antibodies obtained against extensin from carrot roots and tomato cross-react with cell wall proteins from other plants indicating that there are regions highly conserved in the extensin molecules. The same authors reported three types of antigenic determinants or epi- topes in extensin proteins: (1) glycosylated epitope; (2) non-glycosylated epitope of the intact protein and (3)

Cell wall proteins of Prosopis chiknsis

Table 1. Amino acid composition of cell wall proteins

283

Prosopis chiknsis mol % Soybean* Amino acid 180000 126ooO 107000 63 WO SbPRPl ENODZ

HYP

Pro

Asx

Thr

Ser

Glx

GlY

Ala

Val

CYS

Met

Ile

LeU

Tyr

Phe

His

LYS

Arg

Trp

n.d. n.d.

$1 11.5 (1801 2.3

(351 6.8

(107) 23.8

(3771 6.2

(98) 5.0

(79) 4.7 (741 0.8

(13) 0.0

(01 4.4

(78) 7.4

(116) 2.6

(411 2.8

(44) 0.9

(141 4.8

(76) 9.3

(146) n.d.

7.8 (851 11.7 (1291

(:;3 7.1

(781 21.3

(235) 5.7

(631 5.4

(60) 5.5

(61) 0.7

(81 0.0

(01 3.5

(381 6.4

(701

,:; 2.6

(281 2.4

(271 6.2

(681 7.4

(82) n.d.

n.d.

(78;: 10.5

(97) 2.9

(261 6.9

(631 18.6

(1711 4.8

(44) 4.6

(431 6.4

(59) 0.1

(11

:; 3.0

(27)

,z) 5.7

(521

,:;

(4”;“, 8.5

(781

(z+l n.d.

n.d.

14.7

(801 8.9

(471

(:; 3.5

(191 18.7

(102) 3.3

(18) 3.8

(20) 12.3 (671 0.2

(1) 0.0

(0)

t:; 2.5

(141 8.5

(46)

,:: 2.1

(111 13.5 (74) 2.2

(12) n.d.

n.d. n.d.

35.9 44.4

1.2 1.2

0.4 0.4

2.3 0.4

3.9 22.0

0.4 0.4

1.6 0.0

15.2 2.1

0.0 0.0

0.0 0.0

2.4 0.4

3.1 1.2

14.5 7.9

0.0 0.0

0.0 9.5

17.6 9.5

0.4 0.4

0.0 0.0

Numbers in parentheses correspond to the number of residues per molecule. n.d.: Not determined. *From Showalter et al. [ 133.

Table 2. NH,-terminal sequence of cell wall proteins

Protein

(M,)

18OooO X X G N E A L Q E I M X Q 1 Cl 126000 X X Q X Q A L K X I M X X E G G S L L X lO7ooO X X Q I Q A L K X 1 M R R X G 63000 N Y Y Q P HYP T Y K P P E K P X

Amino acids are indicated by single-letter IUPAC nomenclature with the letter X denoting the position of an unidentified residue.

exposed epitopes left after deglycosylation of the protein stance, all four proteins seem to be glycosylated. The

[18]. Some of these epitopes are probably present in the glycosyl residues could be the epitopez for the cross- four cell wall proteins of P. chilensis since all of them reaction of the proteins with the antibodies [19]. A future cross-react with soybean seed coat antibodies. For in- identification of the sugar residues will confirm or rule

284 J. G. RODRIGUEZ and L. CARDEMIL

Table 3. NH,-terminal sequence aligment of the M, 63000 cell wall protein with NH,-terminal sequences found in other proline- rich proteins of other dicotyledonous plants

Amino acids are indicated by single-letter IUPAC nomenclature. The enclosed amino acids indicate the identity with the sequence of P. chilensis protein. The dash means a deletion. The numbers at the left indicate the position of that particular amino acid in relation to the NH,-terminal end of the mature protein. PRPl from ref. [ZO], PRP2 and PRP3 from ref. [IS], NO75 and NO12 from ref. [13] and PR33 from refs [S, 93.

Table 4. Distance matrix for the aligned NH,-terminal se- quences shown in Table 3, the number of amino acid differences

between sequences is indicated

M,63000 PRP2 PRPl PRP3 NO75 PR33 NO12

M,63000 0 PRP2 4 0 PRPI 7 6 0 PRP3 7 8 5 0 NO75 8 6 5 7 0 PR33 9 9 5 7 6 0 NO12 9 8 8 9 10 IO 0

out this possibility, since it is well known that extensins am highly glycosylated with arabinoae. and lesser amounts of galactose [18, 191. In the M, 63 000 protein, homology to the soybean seed coat cell wall proteins seems more evident because the amino acid composition has similar- ities with the derived amino acid composition of proteins encoded in genomic clones of this plant Cl33 and, also, because the NH,-terminal sequence analysis corrobora- tes that this is a protein with homologous sequences to cell wall proline-rich proteins encoded in the soybean genome [ 13, 15, 203.

The results from amino acid composition and NH,- terminal sequence analyses also showed that the other three cell wall proteins could be different proteins from the extensin reported so far. However, it is necessary to point out that the sequences obtained correspond only to the last 15-20 amino acids of the molecule. Therefore, the internal sequences of these proteins, which are unknown, could have homology with some other sequences of proline-rich or hydroxyproline-rich extensin molecules [S, 93. This would also explain the cross-reaction of the three proteins with soybean seed coat antibodies. An antibody raised against deglycosylated extensin could verify if there are non-glycosylated epitopes homologous to extensin.

The higher homology of the M, 63 000 protein to the proline-rich proteins from soybean suggests a close phylogenetic relation between these two legumes. How- ever, the amino acid composition analysis revealed that the M, 63 000 protein had only 14% of proline residues

instead of 35.9 or 44.4 present in the proteins encoded in the SbPRPl or ENODZ genomic clones [13]. In these two soybean proteins, proline residues could also be hydroxyproline residues because this last amino acid does not have a genetic code. However, our amino acid analyses have a serious limitation because there is an underestimation in the hydroxyproline content of the proteins. In our analyses this amino acid co-elutes with aspartic acid/asparagine.

Furthermore, the results of NH,-terminal sequence analysis showed that the M, 63000 protein had two proline-containing peptides with homology to repetitive peptides found in proline-rich proteins present in other angiosperms. This suggests that this protein could have repetitive proline-containing peptides along the molecule, as has been described for all proline-rich proteins [S, 9,13, 15, 201. An antibody raised against one of these two peptides would allow us to confirm whether the M, 63 000 protein is a proline-rich protein and, perhaps, to establish the degree of homology to the two soybean proteins [IS, 203. The same approach would verify whether the pro- teins of M,s 180000, 126 000 and 107000 have any homology with proline-rich proteins present in plant cell walls [S, 91.

EXPERIMENTAL

Biological material. Seeds of P. chilensis (Mol.) Stuntz were collected in Peldehue, near Santiago, Central Chile. Seeds sepd from the legumes, were scarified with coned H,SO, for 10 min to break their dormancy and washed several times with H,O. Seeds were then germinated on wet paper in trays placed in growth chambers at 25” with a 12 hr photoperiod.

Cell wall preparation and protein extraction. Cotyle- dons from 48-hr-old seedlings were wounded with cuts made with a razor blade 48 hr prior to wall prepn [21]. Cell wall prepn and purification was performed in triplic- ate. Cell walls were purified and extracted for proteins, with 0.2 M CaCl, in 2 mM Na$,O, according to the method of refs [22, 231. After extraction, proteins were pptd with 5 vols of cold Me&O. The protein pellet was sepd by centrifugation at 15 000 g and resuspended in 0.1 M Tris-HCl pH 8.8. Proteins were qualified by the

Cell wall proteins of Prosopis chilensis 285

method of ref. [24]. Hydroxyproline content was deter- mined by the method of ref. [25] after hydrolysis of the walls with 6 M HCl at 121” for 12 hr under N,.

Electrophoresis. Cell wall proteins were analysed by SDS-PAGE in a 10% gel as described in ref. [26]. Each well of the gel was loaded with 15-30 pg of proteins. Protein bands were visualized with the silver stain re- agent using the method of ref. [273. Cationic neutral gel electrophoresis analysis of the native proteins was per- formed as described in ref. [28] using 9.7% of poly- acrylamide in the resolving gel. About 10 fig of proteins were loaded in each well of the gel. The electrophoresis was run at 7 mA for 36 hr, using 0.02 M MOPS contain- ing 0.1 M histidine equilibrated at pH 6.8.

Western analyses. After SDS-PAGE or after cationic neutral gel electrophoresis, one-half of the gel was stained to visualize the protein bands. The other half was electro- transferred to nitrocellulose membranes at 130 mA over- night [29] using a MOPS/histidine buffer 1281. After transfer, proteins were identified with a polyclonal anti- body raised against extensin from cell walls of soybean seed coat in a diln of 1: 10000 [30] and a goat secondary antibody in a diln of 1: 25 000 raised against rabbit IgG and conjugated with alkaline phosphatase. Controls us- ing pure carrot extensin, a premune serum and those omitting the first or secondary antibody were run to test the specificity of the antibodies.

Detection of peroxidase actioity. Peroxidase activity was detected in native cell wall proteins separated in cationic neutral gel eletrophoresis using a 0.1% soln of o- phenylenediamine and 0.012% H202 as substrates. The buffer was 0.1 M Na citrate, pH 5 [ 173. The appearance of yellow-orange coloured bands a few min after addition of the reagents was evidence of peroxidase activity. After visualization of the protein bands, the reaction was stopped using successive washes with deionized H,O.

Glycosylation test. Glycosylation of proteins was detec- ted by reaction with periodic acid/dansylhydrazine on gels after SDS-PAGE or electrophoresis of native pro- teins [31]. Protein bands were visualized under UV light.

Amino acid composition. Amino acid composition was determined after prep. purification of each protein. For this, cell wall proteins were sepd by native electrophor- esis, loading 100 pg of protein in each well of the gel and using the same gel concentration and conditions de- scribed for the cationic neutral gel electrophoresis, except that the running buffer used was without histidine. After electrophoresis, proteins were electroblotted overnight on to PVDF membranes according to the procedure of ref. [32]. The transfer was run at 180 mA at 4”. Protein bands were visualized on the immobilon membrane with Coomassie Blue and cut out of the membrane as small ribbons of 4 x 9 mm. Proteins were hydrolysed on the ribbons with 6 M HCI containing a drop of fl-mercapto- ethanol, for 24 hr at 115” under a N, atmosphere. The released amino acids were extracted from the membrane with 200 pl of 30% MeOH containing 0.1 M HCI. After extraction the MeOH soln was lyophilized. The dried powder was resuspended in 0.02 M Na citrate, pH 5.4. The solution (50 ~1) was injected into an amino acid

analyser and quantified by calorimetric determination of the ninhydrin reaction.

NH,-terminal sequences. Proteins were sepd by ~a- tionic neutral gel electrophoresis as described above using the MOPS running buffer without histidine. After electrophoresis, the proteins were electroblotted over- night on to a PVDF membrane at 180 mA at 4” according to ref. [32]. Coomassie Blue staining indicated that transfer of proteins to the membrane was complete. The area of the membrane containing the protein band was cut from the membrane in small ribbons of 4 x 9 mm, each ribbon containing at least 2Opg of protein. The NH,-terminal sequence was determined by Edman de- gradation using an automated sequencer. The phenyl thiohydantoin-amino acid derivatives were identified by reverse-phase HPLC analysis. Comparison of protein sequences was performed with the FASTA alignment analysis computer program [14].

Acknowledgements-We thank James Zobel (Monsanto Corporate Research Protein Biochemistry, U.S.A.), for amino acid composition and NH,-terminal sequence analyses of cell wall proteins. We are grateful to Prof. Joseph E. Varner (Department of Biology, Washington University, U.S.A.) and Dr Gladys Cassab (Department of Plant Biology, University of California, U.S.A.) for their kind gift of extensin antibodies. The technical assistance of Angblica Vega and Ximena Ofiate is ac- knowledged. This research was supported by grants 7,068, Program in Science and Technology Cooperation, Office of Research, U.S. Agency for International Devel- opment, FONDECYT 1140-92 and TRI B 2806-8923 from Universidad de Chile. This work was presented by Jo& Gregorio Rodriguez in partial fulfilment of the requirements to obtain a MS degree at Universidad de Chile. Joti Gregorio Rodriguez was supported by the La Red Latinoamericana de Botinica.

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