P1. Syst. Evol. 186:57-68 (1993) --Plant

Systematics and

Evolution © Springer-Verlag 1993 Printed in Austria

Taxonomic evaluations of the genus Pinus (Pinaceae) based on electrophoretic data of salt soluble and insoluble seed storage proteins

G. PIOVESAN, C. PELOSI, A. SCHIRONE, and B. SCHIRONE

Received August 13, 1992; in revised version December 11, 1992

Key words: Gymnosperms, Pinaceae, Pinus. - Seed storage proteins, electrophoresis, tax- onomy.

Abstract: Electrophoretic studies on seed storage proteins of various gymnosperm species showed that both salt soluble and insoluble protein fractions give taxonomic information. Among species of the genus Pinus evident differences are found between the subgenera Haploxylon (Strobus) and Diploxylon (Pinus). In sect. Strobus the two subsectt. Strobi and Cernbrae are readily distinguished from P. bungeana (sect. Parrya) which appears to fall in an intermediate position between haploxyl and diploxyl pines. Among the species of subg. Diploxylon, "mountain pines" show very similar protein patterns in agreement with their recent speciation (Quaternary).

Electrophoretic analyses on the chemical nature of seed proteins can provide useful taxonomic information (cf. SARKAR & BOSE 1984, KONAREV & al. 1987, COLLADA & al. 1988) which can be applied in evolutionary studies for constructing phylo- genetic hypotheses (e.g., VAUGHAN 1983).

Histological data (LofT 1980, MISRA & GRZEN 1990, PZLOSI & al. 1992) and biochemical evidences (GIFFORD & TOLLEY 1989, FLINN & al. 1991, LAMMER & GIFFORD 1989, PIOWSAN & al. 1991) showed that insoluble cristalloid proteins are important storage sources in Pinus and Picea seeds. Recently, JENSZN & LIxtm (1991) detected the presence of insoluble legumin-like seed proteins in many Pin- aceae but not in Abies. On the other hand, no salt soluble legumin-like proteins had been detected in Pinus and Picea species, whereas their presence had been demonstrated in Ginkgo biloba (JENSEN & BERTHOLD 1989).

To our knowledge, no author has specifically studied the biosystematic impli- cations of the electrophoretic characteristics of Pinaceae storage proteins. Further analysis seemed necessary in view of the importance of seed protein analyses for the taxonomy of Pinus (SCHmONE & al. 1991). This paper reports on new electro- phoretic studies carried out on different pine species, in order to characterize the distinctive bands for each taxon.

58 G. PIOVESAN & al.:

Material and methods

Plant material. Plant species and seed provenances are listed in Table 1. Besides Pinus eight other gymnospermous species were used. Seeds from various sources (field collection and commercial trades) were collected, stored in the dark at 4 °C in 1991, and used in 1992. A pooled sample of 10 seeds was used for each species. The analyses were repeated three times. In Pinus nigra only a detailed screening of intraspecific variability was carried out on 100 individual seeds for each of the four provenances (Calabria, Abruzzo, Carso, Turkey) considered.

Protein extraction. Only mature dry seeds were used for extractions. Seed coats were removed and whole seeds were ground to flour. Extraction and separation into soluble and insoluble fractions were done as described in GIFFORD & al. (1982) with few modifications. This method was adopted because it separates globulins into two major types: salt soluble

Table 1. List of the gymnosperms used in electrophoretic analysis. For Pinus the two subgenera Haploxylon REHDZR (H) and Diploxylon RERDER (D) are indicated

Species Provenances

Ginkgo biloba L. Taxus baccata L. Juniperus macrocarpa SmTH. et SM. Abies alba MILL. Cedrus deodara LOUD. Larix decidua MILL. Picea abies KARST. Pseudotsuga menziesii FRANCO

Pinus brutia TEN. P. bungeana Zucc. P. canariensis SMITH. P. cerebra L. P. densiflora SIEB. et ZtJcc. P. eldarica MEow. P. halepensis MILL. P. leucodermis ANT. P. mugo TURRA P. nigra ARNOLD

P. peuce GRISEB. P. pinaster AITON P. pinea L. P. roxburghii SARGENT P. sylvestris L. P. strobus L. P. thunbergii PARE. P. uncinata MILL. P. waIlichiana JACKSON

BRU BUN CAN CEM DEN ELD HAL LEU MUG NIG

PEU PIR PIN ROX SYL STR THU UNC WAL

a. Italy a. Italy a. Mt Argentario, Italy a. Alpi, Italy a. Pakistan a. Alpi, Italy a. Alpi, Italy a. Siskiyon Cal., USA

a. Turkey a. China a. Canary Isles a. Alpi, Italy a. Japan a. Afghanistan a. Taranto, Italy a. Calabria, Italy a. Trento, Italy a. Turkey b. Carso, Italy c. Calabria, Italy d. Abruzzo, Italy a. Yugoslavia a. Toscana, Italy a. Toscana, Italy a. Pakistan a. Alpi, Italy a. Novara, Italy a. Korea a. Aosta, Italy a. Pakistan

Subgenus D H D H D D D D D D

H D D D D H D D H

Seed storage proteins in Pinus 59

globulins (e.g., legumin-like) and those requiring urea or sodium dodecyl sulphate (SDS) in the extraction buffer for their complete solubility (e.g., cristalloid proteins) (GIFZORD & BEWLEY 1983).

Eleetrophoresis. O n e- d i m e n s i o n a 1 e l e c t r o p h o r e s i s. SDS polyacrylamide gel elec- trophoresis (SDS-PAGE). One-dimensional electrophoresis of the soluble and insoluble proteins under non-reducing (without 2-mercaptoethanol) ( - ME) and reducing (+ ME) conditions was carried out according to LAEMMLI (1970). The 1.Smm slab gels consisted of an acrylamide separation (12.4% T, 3.2% C) and an acrylamide stacking gel (3.5% T, 1.6% C), in the presence of 0.1% SDS. After electrophoresis the gels were stained with 0.05% (w/v) Coomassie Brilliant blue R-250 in 12% (w/v) trichloroacetic acid. Once de- stained, the gels were photographed with T-MAX 100 film. In some cases electrophoretic profiles were examined by an Ultroscan LKB 2202 Laser Densitometer. Proteins molecular weights were estimated by using the Low Molecular Weight Calibration Kit (Pharmacia). The standards employed were: phosphorylase b, 94.0 kDa; albumin, 67.0 kDa; ovalbumin 43.0kDa; carbonic anhydrase, 30.0kDa; trypsin inhibitor, 20.1kDa; a-lactoalbumin, 14.4kDa. In a previous study (SCHIRONE • al. 1991) the SDS-PAGE Molecular Weight Low Range System by Biorad was used where, in a few cases, the molecular weight of these standards differs slightly from those reported by Pharmacia indications.

T w o - d i m e n s i o n a l e l e c t r o p h o r e s i s . (a) SDS-PAGE under non-reducing versus reducing conditions (SDS-PAGE - ME versus SDS-PAGE + ME). The first dimension (1 D) was carried out on proteins extracted under non-reducing conditions in acrylamide rod gels 9 cm long with a diameter of 3.5 mm, under the same conditions as for one- dimensional electrophoresis. After the first dimension the rod gels were incubated in a 0.125 M Tris-HC1 buffer, pH 6.8, containing 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) ME, for 1 hour at room temperature. The second dimension (2 D) was carried out on slab gels under the same conditions as for one-dimensional electrophoresis. Staining was as above. (b) Isoelectric focusing (IEF) versus SDS-PAGE. This analysis was performed according to O'FARRELL (1975). The IEF rod gels were 7 cm long with a diameter of 3.5 ram. The gel mixture contained 4.0% T, 5.4% C, 9M Urea, 2% Nonidet P40, 5% carrier ampholytes by Biorad (pH 3.5-10). Before electrophoresis, reduced protein samples were diluted with an equal volume of a solution containing 10% (w/v) Nonidet P40 and 9 M Urea. It is important to use a percentage ratio of Nonidet P 40 to SDS of at least 8 to 1 (AMES & NIKAIDO 1976). IEF was carried out at constant voltage (250 V). After IEF the rod gels were incubated in the same buffer described in (a). The second dimension was carried out under the same conditions as for one-dimensional electrophoresis. The slab gels were stained first with Coomassie Brilliant Blue as above and then with Silver Stain Kit (Biorad).

Results

Soluble protein gel profiles. All the examined gymnospermous genera can be easily recognized. Ginkgo biloba was characterized by the presence oflegumin-like proteins (JENSEN • BERTOLD 1989). The other genera were distinguished by the position of three major groups of protein bands, which were not reduced in the presence of M E (data not show).

The genus Pinus is characterized by the presence of a clear-cut band between 30 and 36 kDa (A in Fig. 1). Although each species shows different profiles, it is possible to identify two major groups corresponding to the subgenera Haploxylon (32.5-36kDa) and Diploxylon (30-33 kDa). In addition, pines belonging to subg. Haploxylon have a distinctive concentration of bands in the range of 24-27.5 kDa and the appearance of an intense band at 43-48.5 kDa, except for P. bungeana.

6O

Haploxylon Diploxylon

g~ X

G. PIOVESAN 8~ al.:

Haploxylon Diploxylon

Mr

_94

_ 67

_ 43

_ 30

_ 20 .1

_ 14 .4

- M E + ME

Fig. 1. Coomassie blue stained SDS-PAGE profiles of the soluble proteins from seeds of Pinus spp. analyzed under non-reduced ( - ME) and reduced (+ ME) conditions. Molecular weight standards (kDa) are reported on the right of the reduced profiles. For A polypeptides see text. Acronyms of the species are given in Table 1

The same bands were also observed in P. monticola and P. albicaulis (GIFFORD 1988).

Moreover, within subg. Diploxylon it was possible to recognize the so-called "mountain pines from the areas surrounding the Mediterranean" (sensu KLAUS 1989) (P. sylvestris, P. mugo, P. uncinata, and P. nigra). These species are char- acterized by highly similar profiles. This confirmed previous results based on total proteins electrophoresis (SCHIRONE & al. 1991). P. thunbergii and P. densiflora also have a good similarity with the pines above. In contrast, P. leucodermis shows a noticeably different pattern (data not shown; see SCHIRONE & al. 1991). The rel- atively higher variability of "Mediterranean shore and island pines" (sensu KLAUS 1989) is also confirmed (SCHmONE & al. 1991). In particular, soluble proteins supported the evidence of no significant differences between P. eldarica and P. brutia, the former to be considered a subspecies of the latter (CONKLE & al. 1988, SCHmONE & al. 1991).

The intraspecific study carried out on different provenances of P. nigra confirmed the existence of a certain variability in the patterns of soluble proteins as shown in other pines (GIFFORD 1988). In P. nigra the main variations were found in proteins between 30.5-33 and 23-25.5 kDa (Fig. 2). A similar behavior had already been observed in P. pinaster (SCHIRONE & al. 1991: 49, fig. 4) showing the possible use of soluble proteins electrophoresis for provenance studies in Pinus, too.

Finally it is worth noting that the addition of ME does not modify the bands in any species.

Insoluble protein gel profiles. All the examined gymnosperms have insoluble proteins which, by ME addition, can be divided into two major monomer groups

Seed storage proteins in Pinus

a a b b c c d d

M r

61

_ 9 4

_67

_ 4 3

_ 3 0

Fig. 2. Coomassie blue stained SDS-PAGE pro- _ ~.1 files of the soluble proteins from seeds of Pinus

nigra provenances analyzed under reduced con- ditions. Molecular weight standards (kDa) are re-

_ 14.4 ported on the fight of the gel. Letters correspond to Table 1

(large polypeptides L and small polypeptides S) except for Ginkgo biloba and Abies (SCHIRONE & al. 1990, JENSEN & LIXUE 1991).

In Pinus a protein cristalloid inside the protein bodies was reported in both subgenera (LOTT 1980, PELOSI & al. 1992). Electrophoretic analyses in non-reducing conditions demonstrated that such a cristalloid is composed of various protein subunits, the main ones (subunits L-S) being heterogeneous with a molecular weight in the range of 62-54kDa (Fig. 3) (cf. also GIFFORD 1988). Though these bands could be sufficient to separate the two different subgenera, the addition of ME enhances the difference. In fact, in the Haploxylon pines the two groups of monomers are located in a narrower range (L = 35-39.5 kDa; S = 21-23 kDa) than in the Diploxylon ones (L = 33.5-39.5kDa; S = 19.5-23 kDa).

Within the Haploxylon pines a considerable similarity is observed among P. strobus, P. peuce, and P. wallichiana (L = 35-39.5 kDa) whereas P. cembra is clearly distinct (L = 36-39.5 kDa). Comparing our results to those of GIFFORD (1988) it is apparent that P. monticola showed a pattern similar to the first three species while P. albicaulis resembled P. cembra. The electrophoretic profiles of proteins extracted following the method of PAYNE & al. (1981) confirmed these results (see SCHIROyE & al. 1991: 46, fig. 1). Moreover, this extraction method, allowing a higher band resolution, corroborated the specific conservativeness of insoluble proteins (cf. SCHIRONE & al. 1991: 49, fig. 4).

Finally, a protein band appears at approximately 43-48.5 kDa in all the pines analyzed. This band is particularly intensive in all Diploxylon pines and, among the Haploxylon pines examined, only in P. bungeana. However, in the latter subgenus this band is mainly soluble in the salt buffer, except for P. bungeana.

62 G. PIOVESAN & al.:

Haploxylon Oiploxylon Haploxylon Diploxylon

L-S

- r

M r

_94

- - 67

_43

_30

_ 2 0 . 1

_ 1 4 . 4

- ME + ME

Fig. 3. Coomassie blue stained SDS-PAGE profiles of the insoluble proteins from seeds of Pinus spp. analyzed under non-reduced ( - ME) and reduced (+ ME) conditions. Arrows in the profile of P. bungeana show the small dimer ( - ME) and the 1 and s monomer (+ ME). Molecular weight standards (kDa) are reported on the right of the reduced profiles. Acronyms of the species are given in Table 2

Two-dimensional electrophoresis (SDS-PAGE - M E versus SDS-PAGE + ME) shows the organization of the main protein subunits (Fig. 4). These analyses confirmed the existence of two L and S groups of monomers linked by S-S bonds in heterodimeric complexes (L-S subunits) (cf. LAMMER & G~FFORI) 1989).

It is interesting to observe that in P. bungeana a small dimer is also present (37- 38 kDa). In reducing conditions this protein divides into two polypeptides (1 = 21- 23 kDa; s = 15.5-16.5 kDa) indicated by arrows in Figs. 3 and 4. A similar protein has been detected also in P. halepensis, although "s" is scarcely perceptible in this species.

The study on the isoelectric characteristics of the reduced insoluble proteins (IEF versus SDS-PAGE) showed a certain charge heterogeneity of the individual monomers separated according to their molecular weight (Fig. 5); this complexity is typical of globulins from many species (cf. DEP, BYSI-IIP, E & al. 1976, GIFFORD & BEWLEY 1983). Generally, the L monomers are more acidic in subg. Haploxylon than in subg. Diploxylon while S monomers are more acidic in the latter subgenus.

The small dimer detected in P. bungeana seems to be basic in the first monomer (1) and acidic in the second one (s) (arrows in Fig. 5), like in Ginkgo biloba (JZNSEN & BERTHOLD 1989) and in Fagus sylvatica (COLLADA & al. 1991).

Finally, the band detected around 43-48.5 kDa is acidic in all the pines studied and could represent a subunit similar to those detected in Fagus sylvatica, Brassica spp. and Glycine m a x (KAGAWA & HIRANO 1988, MIMOUNI & al. 1990, COLLADA & al. 1991).

Seed storage proteins in Pinus

1D: SDS PAGE - ME M

~iiiiiiiii!i!ii~ii~i!~ii ~ ~ii~!!i~ ~iiii! ~i ~ ~ i ? ~'~i~ ~ ~i~i~ ,~i!ili!i s m : : , ,

2 D

S D S I P A G E

+ M E

BUN (H) HAL (D)

63

Fig. 4. Coomassie blue stained two-dimensional electrophoretograms of the insoluble pro- teins from seeds of Pinus spp. First dimension: SDS-PAGE under non-reduced conditions ( - ME). Second dimension: SDS-PAGE under reduced conditions (+ ME). The mono- dimensional (M) profiles (SDS-PAGE + ME) are reported on the left. Arrows in P. bungeana show the two monomers (1 and s) of the small dimer. Acronyms of the species are given in Table 1

Discussion

It was confirmed that seed proteins are a useful taxonomic tool. Total proteins electrophoresis was proved to be a good method for rapid identification of pine species (SCmRONE & al. 1991). Both salt soluble and insoluble seed storage proteins permit to recognize the genus Pinus from the other examined gymnospermous genera. Moreover, by salt soluble proteins it can be possible to analyze different taxonomic categories including the rank of provenance, while by insoluble proteins the subsection level can be reached.

In Pinus the two subgenera Haploxylon and Diploxylon can be readily recognized. Furthermore, by examining our results and comparing them with those obtained by GIFFORD (1988), three groups of species can be distinguished in subg. Haploxylon: P. cembra-albicaulis, P. strobus-peuce-wallichiana-monticola, and P. bungeana; a clustering corresponding to the most common classifications of Pinus (cf. SHAW 1914, CRIWCHFIELD & L~TTLE 1966, VAN DER BURGH 1973). In fact, the first two pines fall within sect. Strobus LEMMON subsect. Cembrae LOUDON, the next four are included in sect. Strobus subsect. Strobi LOUDON, and P. bungeana in sect. Parrya MAYR subsect. Gerardianae LOUDON.

In sect. Strobus differences between L monomers of the Cembrae and Strobi species agree with the results of DNA analysis (SZMIDT & al. 1988, GOVINDARAJU & al. 1991) supporting an early differentiation of the two subsections. Precedently, analyses of flavonoids (LEBRETON & SARTRE 1982) and physiological tendency to hybridize (WR)cHT 1959, KRIEBEL 1972) had pointed out significant differences between the two subsections. It is aIso worth noting that at the morpho-ecological level (McCuNE 1988, KLAUS 1989) many similarities have been found among the

64 G. PIOVESAN & al.:

S

1D:IEF

BUN (H) PEU (H) CAN (D) UNC (D)

Fig. 5. Coomassie blue stained two-dimensional electrophoretograms of the reduced in- soluble proteins from seeds of Pinus spp. First dimension: IEF; the acidic (pH = 3.5, +) and the basic (pH = 10, - ) end of the gel are indicated. Second dimension: SDS-PAGE. The mono-dimensional (M) profiles (SDS-PAGE) are indicated on the center of the gel. Arrows in P. bungeana show the 1 and s monomers of the small dimer. Acronyms of the species are given in Table 1

pines studied of subsect. Strobi, in spite of the great geographical distance separating these species. Similar considerations can be made for P. cerebra and P. albicaulis (subsect. Cembrae) (cf. VAN DER BURGH 1973, DEBAZAC 1977).

P. bungeana requires a special consideration. As in all the pines of subg. Di- ploxylon (cf. also GIFFORD 1988), in this species the band at 43-48.5 kDa was found to be insoluble in a salt buffer but only in the presence of SDS. In fact, this pine belongs to sect. Parrya being characterized by morphological (e.g., needle numbers per fascicle, mucronate umbo, pinoid or piceoid cross field pitting) and genetic characters (STRAUSS • DOERKSEN 1990, WANG 8~; al. 1991) that suggest an inter- mediate taxonomic position between sect. Strobus of Haploxylon and the Diploxylon pines (VAN DER BUR6H 1973, KLAUS 1980). The presence of a small dimer in P. bungeana, as well as in P. halepensis, correlates with this position. Moreover, this dimer could be interpreted as an ancestral feature since it was also found in Ginkgo biloba (JENSEN & BERTIJOLD 1989). These data agree with the assumption that P. bungeana may be considered as a relict (SzMIDT & al. 1988) of a period (Upper Cretaceous-Early Tertiary) where the two main subgenera of Pinus were not so distinct as they appear today (MILLER 1976).

A higher homogeneity was found among the profiles of Diploxylon species compared with the Haploxylon ones. This confirmed the results of phenolic com- pounds (ERDTMAN ~¢ al. 1966) and total genomic DNA (STRAUSS & DOERKSEN 1990) analyses. However, also in subg. Diploxylon it was possible to differentiate the "mountain pines" from the "Mediterranean" ones, especially on the basis of

Seed storage proteins in Pinus 65

salt soluble proteins. This result was in accordance with the different evolutionary trends proposed for these two groups of pines (VAN DER BURGH 1973, KLAUS 1989, SCnIRONE & al. 1991). In fact, the similar patterns in the soluble proteins of P. sylvestris, P. mugo, P. uncinata, and P. nigra and the similarity between these profiles and those of P. densiflora and P. thunbergii reflected the interfertility of these pines (WRIGHT & GABRIEL 1958) thus justifying their taxonomic position (subsect. Sylvestres LOUDON). Moreover, the considerable similarity among the proteins of "mountain" pines agrees with their likely recent Quaternary speciation (FARJON 1984).

It is well known how the genus Pinus has maintained a considerable homogeneity. Such "conservativeness" which can also be detected at the ecological level (McCuNE 1988), may be attributed to speciation processes mainly due to gene mutations (see DUFFIELD 1952, PEDERICK 1970), in which hybridization plays a major role (cf. PERRY 1987). This evolutionary model seemed to be confirmed by a continuous, though limited variability of the molecular weight of the main storage proteins (cf. GIFFORD 1988). The conservativeness was also confirmed by other biochemical markers as was recently discussed by WATERS & SCHAAL (1991).

The early origin of conifers and of the genus Pinus (fossils from the Jurassic, MIROV 1967) make a detailed reconstruction of their evolution difficult (OHRI & KHOSHOO 1986). Nevertheless, the analysis of seed storage proteins can help to understand important steps of this process by producing more reliable phylogenetic frames. For example, the similarity between the isoelectric characteristics of Hap- loxylon monomers and those of the legumin-like proteins of Ginkgo biloba (cf. JENSEN & BERTHOLD 1989) could be related to the earlier differentiation of subg. Haploxylon (cf. FARJON 1984). In general, due to their conservativeness insoluble proteins can provide useful information for the study of the relationships between sections and subsections of Pinus (cf. CRITCHFIELD & KINLOCH 1986, KARALA- MANGALA & NICKRENT 1989). In this view, our future studies will be extended to other species with special regard to the Parrya group, in order to understand the kind ofphylogenesis - whether by holophyletic or paraphyletic lines - that occurred in Pinus.

We are grateful to Prof. E. GIORDANO for encouraging and facilitating this study and to Prof. S. MAZZOLENI for revision of the English. We gratefully acknowledge the anon- ymous reviewers for their critical comments and helpful suggestions on this paper. We also thank Mr A. PARLANTE for developing photographs.

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Address of the authors: GIANLUCA PIOVESAN, CLAUDIA PELOSI, AVRA SCHIRONE, BAR- TOLOMEO SCHmONE, Department of Forest Environment and Resources, University of Tuscia, Via S. Camillo de Lellis, 1-01100, Viterbo, Italy.

Accepted December 11, 1992 by F. EHRENDORFER