Báo cáo khoa học: "Effects of buffer system pH and tissue storage on starch gel electrophoresis of allozymes in three tropical tree species"

Chia sẻ: Nguyễn Minh Thắng | Ngày: | Loại File: PDF | Số trang:20

Thêm vào BST

Báo xấu

60
lượt xem 1
download

Download Vui lòng tải xuống để xem tài liệu đầy đủ

Tuyển tập các báo cáo nghiên cứu về lâm nghiệp được đăng trên tạp chí lâm nghiệp quốc tế đề tài: "Effects of buffer system pH and tissue storage on starch gel electrophoresis of allozymes in three tropical tree species...

Chủ đề:

Bình luận(0) Đăng nhập để gửi bình luận!

Lưu

Nội dung Text: Báo cáo khoa học: "Effects of buffer system pH and tissue storage on starch gel electrophoresis of allozymes in three tropical tree species"

Original article Effects of buffer system pH and tissue storage on starch gel electrophoresis of allozymes in three tropical tree species PD Khasa WM Cheliak J Bousquet 1 Centre de Recherche en Biologie Forestière, Faculté de Foresterie et de Géomatique, Université Laval, Sainte-Foy, Québec, G 1K 7P4: 2 Département de Biologie, Faculté des Sciences, BP 190, Université de Kinshasa, Zaire; 3 Forest Pest Management Institute, Forestry Canada, PO Box 490, 1219 Queen Street East, Sault-Ste-Marie, Ontario, Canada, P6A 5M7 (Received 7 April 1992; accepted 31 August 1992) Summary — The effects of 16 different electrophoresis buffer pHs, 4 tissue storage conditions and 5 storage times on starch gel electrophoresis of 18 enzymes were determined to design a genetic variation sampling strategy for an isozyme study of 3 tropical tree species, Racosperma auriculi- forme, R mangium, and Terminalia superba. The pH of the buffer systems had a significant effect on the number of putative gene loci and alleles resolved, and the staining intensity of the 18 enzymes assayed. For Racosperma species, 2 buffer systems B (Tris-citrate gel, pH 9.0: lithium hydroxide- 7 borate electrode, pH 8.5) and H (histidine-EDTA gel, pH 7.6: Tris-citrate electrode, pH 7.7) gave 7 the highest average performance in resolving power. All buffer systems yielded poor results for Ter- minalia. Freezing of Racosperma embryos for up to 2 months did not seriously affect enzyme activi- ty. However, freezing cotyledon tissue of Terminalia decreased enzyme activity over a 2-month peri- od. In general, frozen tissues either with or without extraction buffer, were consistently better than frozen tissues with extraction buffer and DMSO. Three classes of enzymes were defined, based on their stability under the standardized storage conditions in vivo. Using the best buffer systems (B 7 and H and tissue storage conditions (To or T 42, 43, and 32 zones of activity were resolved for R ) 7 ), 1 auriculiforme, R mangium, and T superba, respectively. Genetic inference of enzyme variants was made for 31 and 32 putative gene loci in R auriculiforme and R mangium, respectively. Mean num- ber of putative alleles per locus was 3.0 for R auriculiforme and 2.4 for R mangium. buffer system pH / starch gel electrophoresis / allozyme genetic inference / plant material storage / Racosperma / Terminalia / tropical tree Résumé — Effets du pH du système de tampons et de la conservation des tissus en électro- phorèse sur gel d’amidon d’allozymes chez 3 espèces d’arbres tropicaux. En vue de planifier une stratégie d’échantillonnage de la variabilité génétique de 3 espèces d’arbres tropicaux, Racos- * Correspondence and reprints
mangium et Terminalia superba, les effets de 16 différents pH de tampons perma auriculiforme, R d’électrophorèse (tableau /), de 4 conditions de conservation des tissus et de 5 durées de conserva- tion ont été évalués pour l’électrophorèse sur gel d’amidon de 18 enzymes. La résolution du nombre de loci et d’allèles présumés possibles ainsi que l’intensité de coloration des 18 enzymes étaient in- fluencées de manière sensible par le pH des systèmes de tampons. Pour les espèces de Racosper- ma, deux systèmes de tampons, B (Tris-citrate, pH du gel 9.0: hydroxyde de lithium, borate- pH de 7 l’électrode 8,5) et H (histidine, EDTA, pH du gel 7,6: Tris-citrate pH de l’électrode 7,7) ont donné le 7 meilleur pouvoir moyen de résolution (fig 1-11, tableau II). Tous les systèmes de tampons ont entraî- né des résultats insatisfaisants chez Terminalia. La congélation des embryons de Racosperma pour plus de 2 mois n’a pas affecté sérieusement l’activité enzymatique. En revanche, la congélation des cotylédons de Terminalia au-delà de 2 mois a diminué l’activité enzymatique. En général, les tissus congélés avec ou sans tampon d’extraction, donnaient constamment de meilleurs résultats que les tissus congélés avec le tampon d’extraction supplémenté de DMSO (fig 12). Trois classes d’enzymes ont été définies, sur la base de leur stabilité sous les conditions in vivo standardisées (tableau III). En utilisant les meilleurs systèmes de tampons (B et H et conditions de conservation (T ou T 42, 01 ), 77 ) 43 et 32 zones d’activité étaient séparées respectivement pour R auriculiforme, R mangium et T su- perba. L’inférence génétique de 31 et 32 loci présumés a été conduite pour R auriculiforme et R man- gium, respectivement (fig 13-17). Le nombre moyen d’allèles présumés par locus était de 3,0pour R auriculiforme et de 2,4 pour R mangium (tableau IV). pH de tampons d’électrophorèse sur gel d’amidon / inférence génétique d’allozymes / conser- vation du matérial végétal /Racosperma /Terminalia / arbre tropical INTRODUCTION 57% of proteins differing by single amino acids can be separated on a single gel. Their study implies that proteins with differ- Isozyme analysis has been used over the ent amino acid sequences will have identi- past 2 decades to investigate the genetics cal electrophoretic mobility in 50% of all ≈ of a large number of organisms, from fruit comparisons using single gel. However, a flies and wild herbs to humans (Nevo, the resolving power of electrophoresis 1978). One of the most widely used proce- could be enhanced by running a sequence dures for studying gene-based variation is of gels at different pH values (sequential through isozyme variation in starch gel electrophoresis), because proteins not electrophoresis. This technique has been separated at one pH may be separated at especially powerful in the study of popula- another. Sequential electrophoresis is tion genetics of forest tree species (Mitton, presently one of the best methods for dis- 1983; Hamrick and Loveless, 1989; El- tinguishing among protein molecules Kassaby, 1991). Powell (1983), Hartl and (McLellan and Inouye, 1986). In the same Clark (1989), Lewontin (1991) and others way, isoelectric focusing (IEF), polyacryla- pointed out that the validity of estimates of mide gel electrophoresis (PAGE) and 2- polymorphism based on electrophoresis is dimensional (2-D) gel electrophoresis can questionable: the amount of polymorphism be used to study the polymorphism of en- may be underestimated because conven- zymes (McLellan et al, 1983; Görg et al, tional electrophoresis fails to detect many 1988a, 1988b), but the procedures may be amino acid substitutions. McLellan and difficult to apply to a large number of indi- Sherman (1991),using non-denaturating viduals required for population genetics. electrophoresis, reported that from 40-
Isozyme analysis requires material suit- 1984). Liengsiri et al (1990a) reported that, able for enzyme extraction. Seeds of forest for certain tropical species such as Ptero- trees, especially gymnosperms, have been carpus macrocarpus and Dalbergia cochin- extensively used for electrophoretic sur- chinensis, storage of seed tissue in a refrig- veys of genetic variation and for the analy- erator (4 °C) or a freezer (-20 °C) severely sis of mating systems (Cheliak and Pitel, reduced enzyme activity. Cryogenic meth- 1984; Adams and Birkes, 1991; El- ods with liquid nitrogen and lyophilization Kassaby, 1991).The advantages of using have been used for storage (Hamrick and seed material for isozyme analysis are, Loveless, 1986; Santi and Lemoine, 1990) firstly, that storage conditions tend to be but these facilities are not always available simpler than for other tissue types, second- in developing countries. Thus, for tropical ly, that relatively little space is required to species, the challenge is to determine tis- store large numbers of genotypes, and sue storage, and enzyme extraction condi- thirdly, that newly germinated embryos are tions which permit long-term storage and relatively free of substances inhibiting en- optimum resolution in the gels. zyme activity (Loomis, 1974; Rhoades and This paper reports the effects of 16 val- Cates, 1976). However, for tropical spe- of electrophoresis buffer pH, 4 tissue ues cies, seed collection is a major problem storage conditons, and 5 storage times on and it may prove difficult to obtain ade- the capacity of starch gel electrophoresis quate samples (Gan et al, 1981; Liengsiri to resolve enzymes from 3 tropical tree et al, 1990a; Wickneswari, 1991). species, Racosperma auriculiforme (Cunn ex Benth) Pedley (formerly Acacia auriculi- Tissue storage, optimum stage of germi- formis), R mangium (Willd) Pedley, comb nation, and subsequent storage of extracted nov (Formerly A mangium), and Terminalia enzymes need to be determined for each superba Engler and Diels. The first 2 be- species (Vigneron, 1984; Pitel and Cheliak, longing to the family Leguminosae, are 1986a, 1988; Pitel et al, 1989). Methods of used as fast-growing trees for fuelwood protein extraction, electrode and gel buffer plantations while the latter, a member of preparation, as well as enzyme staining rec- the family Combretaceae, is used for tim- ipes for temperate tree species are well ber production in Zaïre. Genetic inference characterized (Conkle et al, 1982; Cheliak of enzyme variants is also presented for and Pitel, 1984; Pitel and Cheliak, 1984, the first 2 species. 1986a; Bousquet et al, 1987). More recent- ly, Kephart (1990) has reviewed the techni- cal aspects of plant enzyme electrophore- MATERIALS AND METHODS sis. However, only a few of these procedures have been developed for tropi- cal species (Vigneron, 1984; Hamrick and Source of plant material Loveless, 1986; Liengsiri et al, 1990a, 1990b; Wickneswari and Norwati, 1991). As many samples are prepared and analyzed Bulked seed collections of 3 tropical tree spe- simultaneously, pre-treatments that pro- cies: R auriculiforme (exotic to Zaire), R man- gium (exotic to Zaire), T superba (indigenous to mote uniform germination of seed samples Zaïre) were used in this study. The seeds of 13 have also to be determined. With temperate populations of A auriculiforme and 13 popula- species, once the enzymes have been suc- tions of R mangium were provided by the Com- cessfully extracted and protected, they can monwealth Scientific and Industrial Research be stored for extended times at -70 °C with Organization (Australia), the Centre de Coopéra- little loss of activity (Cheliak and Pitel, tion Internationale en Recherche Agronomique
From preliminary studies, cotyledon tissue of pour le Forêt Développement, Département (CIRAD-FORÊT) (Congo) Terminalia superba was found to be better than and the Service Na- radicle tissue for extracting enzymes. Therefore tional de Reboisement-Centre Forestier de Kin- zono (Zaire) and those of Terminalia were collect- = 100 mg of cotyledon tissue was used with 50 μl complex vegetative extraction buffer (0.05 M ed at Luki Biosphere Reserve (lat 5°37’S, long 13°6’ E, alt 350 m) in Zaïre from 5 parent trees. boric acid, 2% (v/v) tergitol, 2% (w/v) polyethy- More details on the origin of the Racosperma lene-glycol (PEG 20 M), 7% (w/v) polyvinylpyr- rolidone (PVP 40 M), 1% (w/v) PVP 360 M, 50 seeds are given elsewhere (Khasa et al, 1993a). mM ascorbic acid, 0.4 mM NAD, 0.1% (w/v) BSA, 0.2 mM pyridoxal-5’-phosphate (P-5-P), 0.3 M sucrose, 12 mM cysteine-HCl, 1.3% (v/v) Seed germination β-mercaptoethanol). Seeds of R mangium were pretreated by im- Electrophoresis mersing 3 vol seeds in 10 vol 100 °C water until cool (12-24 h). Seeds of R auriculiforme and Prior to electrophoresis, both fresh and previ- T superba were chemically scarified with H 4 SO 2 ously frozen embryos or cotyledons were ho- 95-98% (v/v) for a period of 15 or 30 min, then mogenized with a power-driven Teflon rotating rinsed under running tapwater for 15 min (Kha- tissue grinder. Crude homogenate was ab- sa, 1992, 1993). Pretreated seeds were germi- sorbed onto 1 x 14 mm wicks cut from Whatman nated on Kimpak K-22 media (cellulose paper No 3 filter paper and loaded into 12.5% (w/v) from Kimberly-Clark, WI, USA) in clear seed starch gels prepared from hydrolyzed starch germination boxes (28 x 24 x 6 cm dimension, (Connaught Laboratories, Willowdale, Ontario, from Spencer-Lemaire Industries, Edmonton, Canada). Each gel contained 20 samples of Alberta, Canada) as described by Wang and each of the populations of the 3 species and Ackerman (1983). The Kimpaks were initially electrophoresis was repeated twice. moistened to saturation point with distilled wa- ter. Germination was in Conviron G30 germina- Two different running buffer systems (B or tors (Controlled Environments, Winnipeg, Mani- H) according to Cheliak and Pitel (1984) and toba, Canada) with an 8 h-16 h photoperiod Liengsiri et al (1990b) were tested with 16 pH (day-night), 30 °C-20 °C temperature regime conditions ranging from pH 5.6-9.3 (table I). (day-night), and conditions of high humidity The electrophoresis was carried out to reveal (85% RH). Light was supplied from fluorescent the activity of 18 enzymes: acid phosphatase lamps at an intensity of 12 μmE.m . -1 .s -2 (ACP, EC 3.1.3.2), aconitase (ACO, EC = 4.2.1.3), aldolase (ALD, EC 4.1.2.13), alkaline phosphatase (ALP, 3.1.3.1),aspartate amino- transferase (AAT, EC 2.6.1.1), diaphorase Effect of electrophoresis buffer pH (DIA, EC 1.8.1.4), esterase-colorimetric (EST- c, EC 3.1.1.1), glucose-6-phosphate dehydrog- Enzyme extraction enase (G6P-DH, EC 1.1.1.49), isocitrate dehy- drogenase (IDH, EC 1.1.1.42), leucine amino- Newly germinated embryos of Racosperma peptidase (LAP, EC 3.4.11.1), malate excised from the seed coat and dehydrogenase (MDH, EC 1.1.1.37), malic en- were were placed individually in 0.5-ml conical polystyrene zyme (ME, EC 1.1.1.40), nicotinamide adenine dinucleotide dehydrogenase (NADH DH, EC sample cups (Elkay Products, Shewbury, MA). A small quantity (50 μl) of seed extraction buffer 1.6.99.3), phosphoenolpyruvate carboxylase (30 mM Tris, 5 mM citric acid, 0.4 mM β- (PC, EC 4.1.1.31),6-phospho-gluconate dehy- nicotinamide adenine dinucleotide (NAD), 0.2 drogenase (6-PGDH, EC 1.1.1.44), phosphog- mM β-nicotinamide adenine dinucleotide phos- lucose isomerase (PGI, EC 5.3.1.9), phosphog- phate, sodium salt (NADP), 1 mM ascorbic acid, lucomutase (PGM, EC 5.4.2.2), shikimic acid 1 mM ethylenediaminetetraacetate-disodium dehydrogenase (SDH, EC 1.1.1.25). These en- (EDTA), 0.1% (w/v) bovine serum albumin stained following Cheliak and Pitel were zymes (BSA), pH adjusted to 7.0 with 1 M citric acid) and Liengsiri et al (1990a) with minor (1984) added to each cup. modifications. was
Effect of tissue storage conditions and freezing periods In this experiment, 4 tissue storage conditions and 5 storage times were examined. The stor- age conditions were: T (frozen tissue without 1 extraction buffer), T (frozen tissue immersed in 2 50 μl of extraction buffer), T (frozen tissue im- 3 mersed in 20 μl of dimethyl sulfoxide (DMSO) cryoprotective agent (see Kephart, acting as a 30 μl extraction buffer), T (frozen tis- 4 1990) + sue immersed in 30 μL DMSO + 20 μl extraction buffer). To (fresh tissue) was considered as the standard. For T the 5 storage times tested , 4 -T 1 were: 1 wk, 1, 2, 3 and 6 months. Before grind- ing the samples, 50 μl of sample extraction buf- fer was added to the treatments To and T and 1 frozen tissues were allowed to thaw. Extraction buffers and methods used in this experiment were those described above. For each combina- tion of species, tissue storage conditions, and storage times, twenty samples were then run fol- lowing protocols described in Experiment 1 by using B and H buffer systems, which proved to 7 7 be the most reliable (see below). Enzyme activi- ty was also assessed visually using a 6-step score as above, where 0 means no enzyme ac- tivity and 5 is the standard corresponding to the enzyme activity in fresh tissue. For each combi- nation of tissue, storage condition and storage time, the scores were also averaged across the enzyme zones and expressed in percentage rel- ative to the standard (T to define the average ) 0 remaining percentage of enzyme activity (AR- PEA), which was used to identify the best tissue storage condition and storage time. Genetic inference of enzyme variants in Racosperma intensi- resolving power and the The staining evaluated for each enzyme and pH con- ty were Using the best buffer systems (B and H and bad resolu- ) 7 a 6-step score (0 7 dition by using = tissue storage conditions (To or T presented tion, 1 poor, 2 average, 3 good, 4 very ) 1 = = = = herein (see below), genetic inference of enzyme good, 5 excellent) and by estimating the mi- = variants for Racosperma species was performed gration distance of the common allozyme (stan- dard) within a zone of activity compared to the by comparison with results previously reported in these species (Moran et al, 1989a, b) and by total distance that the buffer front migrated (R ). f the examination of the active subunit composi- For each pH buffer system, the scores were av- tion of each enzyme. At least 60 seeds from eraged for all the enzyme zones assuming 5 as each of 13 different populations for each Racos- a maximum score and expressed as a percent- perma species were analysed for the inference age of the maximum score in order to identify of allozymes. Putative allelic identity was con- the best buffer system.
H were the best buffer sys- and firmed across populations within species by run- 7 78 BB , ning different populations on the same gel. When displayed poor resolution and tems but more than one zone of activity was detected for a weak staining intensities for most of the particular enzyme, the most-anodally-migrating enzymes tested. of activity (nominally putative locus) was a zone designated as 1 and any other numbered accord- ing to decreasing mobility. Within each putative Acid phosphatase (ACP) gene locus, the most anodal allozyme (nomically a putative allele) was assigned the number 1, 2 When analysed with the B buffer system, 5 the next most anodal and so on. R is the fa was 3 zones of activity were observed for each mobility of the various allozymes. For each spe- of the 3 species. The R were 0.32, 0.16, ’s f cies, the mean number of putative alleles per lo- cus (A including the null alleles, was calculated ), s and -0.03 for both Racosperma species. following the formula A 1/m &i where m is the s Sigma;a The third zone (Acp&num;3) was stained on the = number of putative loci scored, and a is the num- i cathodal strip. For T superba, the R were ’s f ber of putative alleles observed at locus i. Be- 0.32, 0.22, and 0.12. Using the B buffer 7 cause of the small sample size used and the system, all 3 zones migrated anodally in poor resolution obtained in Terminalia superba, Racosperma. genetic inference of enzyme variants was not conducted in this species. RESULTS Effect of electrophoresis buffer pH The 18 enzyme systems used in this study displayed 42, 43, and 32 zones of activity for R auriculiforme, R mangium, and T su- perba, respectively (see below for descrip- tion). The effect of buffer pH on some of these zones is shown in figures 1-10. In general, clear and consistent zones of ac- tivity were observed for both Racosperma species while poorly resolved zones were typical for T superba. The buffer systems resulting in highest levels of enzyme activity and resolving power across the different enzyme sys- tems assayed for the 3 species are shown in table II. Certain enzymes such as ACO, ALP, MDH, and SDH proved to have broad pH range tolerance, particularly for T superba. On the basis of the averaged scores in percentage, B and H were the 7 7 best buffer systems among the different B and H buffer systems assayed for both Ra- species (fig 11).For T superba, cosperma
Aconitase (ACO) zone probably represents a single locus with a total of 3 single-banded phenotypes Using the H buffer system, 2 zones of ac- while the variants appeared as single- 7 tivity having R of 0.50 and 0.38 were de- banded phenotypes in the more cathodal ’s f tected for both Racosperma species. The zone (Ald&num;2). For T superba, one clear anodal achromatic where- and consistent band with R of 0.22 could f more zone was the second developed a bluish back- usually be observed. as ground. Only one blue background zone with R of 0.64 was present for T superba, f Alkaline phosphatase (ALP) when analysed with H buffer system. 4 A singe zone of activity was observed with Aldolase Rof 0.18 and 0.10 for Racosperma spe- (ALD) ’s f cies and T superba, respectively, when an- Using the H buffer system, 2 zones alysed with H buffer system. A 1.5-mm 8 6 stained for both Racosperma species with slice is preferred because thinner gel slic- R of 0.30 and 0.12. The more anodal ’s f es showed weak staining of bands.
Aspartate aminotransferase (AAT) R auriculiforme and R mangium respec- tively, and 0.31 for T superba. Using the B buffer system, 3 zones of ac- 7 tivity were detected. The R were 0.35, ’s f lsocitrate dehydrogenase (IDH) 0.27, 0.09 for R auriculiforme, 0.27, 0.23, 0.09 for R mangium and 0.36, 0.22, 0.18 buffer system, one zone of the Using 7 H for T superba. With the B buffer system, 5 observed with R of 0.40 and activity ’s f was the most cathodal zone (Aat&num;3) for Racos- 0.38 for Racosperma species and T super- perma species was close to the origin of ba, respectively. the gel and was indistinct and unscorable. This suggests a zwitterion, which has its Leucine aminopeptidase (LAP) isoelectric point close to the pH condition used. Two zones of activity were detected when analysed with the B or B buffer system. 8 7 Diaphorase (DIA) The R were 0.39 and 0.32 for both Racos- ’s f perma species. When analysed with any With the B buffer system, 3 zones of activ- 7 other buffer system, the 2 zones were close evident. However only 2 zones ity were and indistinguishable. Two poorly resolved could be consistently scored. The R/s of zones were also observed for T superba. the first 2 zones were 0.32 and 0.24 for both Racosperma species. For T superba, Malate dehydrogenase (MDH) the 2 zones were indistinct and impossible to score. Using the H buffer system, 3 zones of ac- 7 tivity (Mdh&num;1, Mdh&num;2, Mdh&num;3) could usual- Esterase-colorimetric (EST-c) ly be observed, with R of 0.35, 0.29, 0.06 ’s f for the Racosperma species, and 4 zones With the B buffer system, 7 and 8 zones 7 R of 0.42, 0.41, 0.15, -0.06 for T su- with ’s f of activity were observed for R auriculi- perba. For the Racosperma species, the forme and R mangium respectively, with most anodal (Mdh&num;1) stained intensely, Rof 0.60, 0.50, 0.41, 0.29, 0.21, 0.14, ’s f the next most anodal (Mdh&num;2) was often 0.07 and 0.60, 0.50, 0.45, 0.40, 0.34, 0.29, obscured by the excessively heavy stain at 0.21, 0.14. When B and B buffer systems 3 4 Mdh&num;1 so that slight mobility differences at were used, the 3 least anodal zones Mdh&num;2 may often be difficult to detect. A (Est&num;6, Est&num;7 and Est&num;8) stained on the putative interzone was apparently present cathodal strip and the resolution was bad. in population of R auriculiforme between T superba showed 4 zones of activity for Mdh&num;2 and Mdh&num;3 (Khasa et al, 1993a). EST-c but they were poorly resolved. The third zone (Mdh&num;3) displayed faint bands in Racosperma species. For T su- perba, the first 2 zones were comigrating. Glucose-6-phosphate dehydrogenase While Mdh&num;1, Mdh&num;2, and Mdh&num;3 stained (G6P-DH) on the anodal gel strip, the fourth zone was stained on the cathodal strip when the When analysed with the H buffer system, 7 H buffer system was used. Using the H single zone of activity was evident in the 8 1 a buffer system, Mdh&num;4 was very close to 3 species. Staining intensity and resolving the origin of the gel but migrated anodally power were poor for T superba. The R of ’s f 0.36 and 0.40 for for the observed T superba. zone were
Malic enzyme Racosperma species. Under low pH condi- (ME) zones could be scored. The tions, only 2 Two zones of activity with R of 0.39 and for R auriculiforme and R mangium ’s f ’s f R 0.12 were observed for Racosperma spe- were 0.52, 0.38, 0.33 and 0.46, 0.38, 0.33 cies and T superba when analysed with respectively. For T superba, 2 zones with the H buffer system. 7 R of 0.48 and 0.27 were detected. ’s f Nicotinamide adenine dinucleotide 6-phosphogluconate dehydrogenase dehydrogenase (NADHDH) (6-PGDH) Three zones of activity were observed for When analysed with the H buffer system, 7 the Racosperma species, but the most 2 zones with R of 0.40 and 0.33 were ’s f anodal zone stained inconsistently and found for the 3 species. At high pH condi- therefore could not be scored. The most tions, only the second zone was detected cathodal zone (Nadhdh&num;3) was faint. With for T superba. the B buffer system, the R of Nadhdh&num;2 7 ’s f and Nadhdh&num;3 were 0.26, 0.21 and 0.23, Shikimic acid dehydrogenase (SDH) 0.19 for R auriculiforme and R mangium, respectively. No enzyme activity was de- With the H buffer system, a single zone of 2 tected for T superba. activity with an R of 0.41 was found for f Racosperma species. Two zones stained Phosphoenol pyruvate carboxylase strongly with R of 0.35 and 0.23 for T su- ’s f (PC) perba. When analysed with the B buffer system, 5 1 zone of activity with R of 0.17 was ob- f Effect of tissue storage conditions served for the 3 species. As staining is and storage times strong but washes off the gel quickly, this enzyme must be scored at the optimal time. Based on their sensitivity to cold storage after different tissue storage times and fol- lowing 4 storage conditions, we have de- Phosphoglucose isomerase (PGI) fined 3 classes of enzyme (table III): 1) high stability enzymes (HSE) which include With the B buffer system, 2 zones of ac- 7 AAT, EST, 6-PGDH, PGI, and PGM for tivity (Pgi&num;1 and Pgi&num;2) were observed. which between 67-100% of the enzyme The R were 0.30 and 0.18 for both Ra- ’s f activity remained for the best treatment af- cosperma species. When the B buffer 3 ter 6 months offreezing; 2) medium stabili- used, the second zone Pgi&num;2 was too was ty enzyme (MSE) including ACP, ALP, G6- close to the origin to be scored. Only 1 PDH, LAP, and MDH (recovery between zone of PGI activity was detected for T su- 33-66%); 3) low stability enzyme (LSE) in- perba with an R of 0.30. f cluding ACO, ALD, DIA, IDH, PC, ME, NADHDH, and SDH (recovery < 33%). The Phosphoglucomutase (PGM) average recovery for the 18 enzymes as- When analysed with the H buffer system, sayed (fig 12) indicated that embryos of 7 3 zones of activity were observed for the Racosperma may be stored in a freezer for
2 months and still show an average re- the authors upon request). The storage = covery > 60% whereas an average recov- conditions T and T resulted in the high- 1 2 ery of≈ 50% was observed after 1 month est enzyme activity on average with appar- of cotyledon tissue freezing for T superba. ently no significant difference whereas ad- After 2 months of freezing for T superba, dition of DMSO (T and T decreases 3 ) 4 the average recovery was < 50% and the enzyme activity markedly. Preliminary results were highly variable from one en- studies indicated that weaker banding pat- zyme to the other as indicated by the large terns were also obtained when glycerol standard deviations (results available from was used as a cryoprotectant.
Genetic inference of enzyme variants Putative gene loci and allozyme variants in presented in table IV. Racosperma are Thirty-one and 32 putative gene loci were inferred for R auriculiforme and R man- gium, respectively. Of these loci, 28 and 25 were polymorphic for R auriculiforme and R mangium, respectively. The average numbers of putative alleles per locus were 3.0 and 2.4 for R auriculiforme and R man- gium, respectively, and the respective numbers of putative alleles per polymor- phic locus were 3.4 and 3.1. Zymogram phenotypes for monomorphic or polymor- phic putative isozyme loci in Racosperma are illustrated in figures 13-17. In the present study, inheritance of enzyme vari- not inferred in Terminalia but has ants was been for loci proposed by Vigneron some (1984). DISCUSSION Starch has been fre- gel electrophoresis used in a successful manner for quently surveys of isozyme variation. However, this technique is susceptible to quantitative inaccuracy and irreproducibility (Gordon et al, 1988; Walters et al, 1989; Kephart, 1990) because of inconsistent staining, un- interpretable inheritance patterns, or other problems. Many factors may be responsi- ble for these problems (Kephart, 1990). Some of these factors include the nature of the tissue and type of storage conditions, protocols for enzyme extraction, starch gel preparation and electrophoretic conditions, gel and electrode buffer characteristics, staining and genetic interpretation of en- and electrode buffers) on the resolving zyme phenotypes. The production of unre- power and staining intensities of electro- liable or reliable results depends much morphs. Follwing a conservative approach more on the skills of the experimenter. by altering the pH of the buffer systems in increments of 0.4 pH units (Kephart, In our first experiment, we have investi- ≈ 1990), it was possible to identify the best gated the effect of buffer pH (B and H gel
pH conditions of buffers for each of 18 en- such buffer systems and pH conditions as account for most of these differenc- zyme systems (table II). Using 12 gel- likely electrode buffer systems, 22 out of 40 en- es. In addition, more polymorphic loci have zyme systems assayed in Vitis species been observed in this study, especially for were successfully resolved (Walters et al, R mangium which has been described as 1989). However, a compromise must be al, genetically depauperate (Moran et struck between resolving power in terms of 1989b). the number of zones scored, and keeping The resolution of extracts of T superba the whole process economical to avoid un- poor when compared to Racosperma, was necessary expenditure of chemicals and even with B and H buffer systems. But 7 7 time. Using only H and B running buffer the number of zones resolved in this study systems in 3 tropical tree species (Ptero- (32) was substantially greater than that re- carpus macrocarpus, Dalbergia cohcinchi- ported by Vigneron (1984), who scored 4 nensis, and Pinus kesiya), a satisfactory zones for EST-c, 3 zones for AAT, 2 zones number of zones of enzyme activity was for LAP, 2 zones for PGM, 1 zone for PGI, obtained by staining 15 enzyme systems 2 zones for ACP, and 2 zones for MDH. A (Liengsiri et al, 1990b). Likely, from our re- browning effect was also observed in Ter- sults of average scores (fig 11),we recom- minalia homogenates when the radicle mend the use of B and H buffer systems 7 7 was used instead of cotyledon tissues, for Racosperma species to stain satisfac- suggesting a loss of enzyme activity result- torily the 18 enzymes used in this study. ing from phenoloxidase products (see Ke- phart, 1990). It is obvious that buffer sys- to results in table According presented tems developed for one species may have IV, the number of putative loci and alleles to be modified depending on the tissue resolved was greater than that previously and enzyme of interest for another spe- reported by Moran et al (1989a, b) in Ra- cies. In that case, the divide and conquer cosperma. Using Tris-citrate and morpho- approach (Kephart, 1990), where a wide line-citrate buffers and 18 enzymes, Moran variety of buffer systems under different et al (1989b) scored 30 loci in R mangium, pH conditions and enzyme combinations as compared to 32 putative loci from 18 must be applied with prior experimentation enzymes in this study. From 12 enzyme on different extraction buffers and different systems assayed in R auriculiforme, Mo- volume to various plant tissue ratios. ran et al (1989a) scored 19 loci, as com- Proteins, which are zwitterions, carry pared to 31 putative loci from 18 enzymes positive and negative charges. Their net in this study. The mean number of putative charge, and thus their migration depends alleles per locus (A was 3.0 and 2.4 re- ) s on the pH of the buffer system. For exam- spectively for R auriculiforme and R man- ple, using the B and B buffer systems re- gium in this study, as compared to pub- 3 5 lished estimates of 2.5 and spectively, even with longer run times, 1.4, respectively (Moran et al, 1989a, b). Differ- Pgi&num;2 and Aat&num;3 zones for Racosperma species were close to the origin of the gel ent sampling and analytical procedures
and did not migrate because at their isoe- cause this organic compound produces weaker ionic strength even when it in- lectric points, these isozymes are electri- creases molarity. Hence, buffers used to cally neutral and do not migrate. For some prepare gels are more sensitive than the pH conditions (B B B and H 3 zones ,, 345 ), 3 electrode buffer where acidic or basic ti- of EST-c (Est&num;6, Est&num;7, Est&num;8), 1 zone of trant may be used to adjust pH. For en- ACP (Acp&num;3), and a few bands of ALP mi- zymes of broad pH range (tolerance), the grated cathodally for the Racosperma spe- composition and molarity of buffer solu- cies, as did Mdh&num;4 for T superba. At high tions are critical in improving resolution. pH conditions, these electromorphs mi- grated anodally. Therefore the pH affects Our results showed that the length of the charge and separation of enzymes al- time that plant tissues could be refrigerat- lowing the possibility of increasing the ed or frozen and still retain enzyme activity number of zones of enzyme activity. It is was variable between species, tissues, relevant to note that the R values and f and enzymes. For high stability enzymes, numbers of bands may also change if dif- it was possible to detect at least 60% of re- ferent electrode and gel buffers and/or pH maining enzyme activity for the best tissue conditions are used. This point is well illus- storage condition after a 6 month freezing trated in this study for enzymes ALP, period of Racosperma embryos. Other re- MDH, PC and SDH (see R values of en- f searchers reported a great loss of activity zyme description in results and figures 13- and indistinct banding pattern for several 17). On the other hand, most allozyme sur- enzyme systems from other plant species veys use gels with pH values close to the after short periods of storage (see Kephart, isoelectric point of the proteins, so that a 1990; Liengsiri et al, 1990a). On the other given difference in charge of the mole- hand, after 2 months, freezing cotyledon cules produces a greater difference in mo- tissue of T superba yielded unsatisfactory bility than if a pH far from the isoelectric enzyme activity. Heterogeneity was also point was used (McLellan, 1984). observed from one enzyme to the other, some being more stable than others re- The way to achieve the desired pH is garding tissue storage in frozen conditions. another concern for some enzymes. For In vitro experimentation would be neces- example, we did not obtain good resolu- sary to confirm the 3 classes of enzyme tion for EST-c even with the B and B buf- 7 8 stability defined here in vivo. fer systems when NaOH (0.1 N) was used to adjust the pH, as suggested by Liengsiri The storage conditions T and T were 2 1 et al (1990a). This could be explained by the best, indicating that the possible stabili- the fact that NaOH increases the ionic zation effect of DMSO during long periods i &2z Sigma; strength (Ic= 1/2 c where z is the va- of freezing is not evident for these species. , i i lency of the ion and ci its concentration) in Apparently, its addition only served to di- the buffer. Buffers of high ionic strength re- lute the extract, thus decreasing banding sult in greater heat production (Andrews, intensity. Smaller quantities of cryoprotec- 1981; Kephart, 1990). Heating tant would be necessary to test this hy- see was also observed when the differential pH be- pothesis. On the other hand, a net positive tween electrode and gel buffers was large. effect of DMSO to stabilize extracts during Gelfi and Righetti (1984) also reported a long periods of ultra-cold storage was re- relationship between the pH gradient, buf- ported in other species, when tissues were fering power, and ionic strength. There- homogenized prior to storage (Kephart, fore, we recommend that the pH of B gel 1990). It is likely that the potential stabiliza- buffers be adjusted by using 1 M Tris be- tion effect of DMSO is more dependent on
extraction procedure and technique of stor- the seed coat of Racosperma species ing age than on species: perhaps with whole has also yielded uniform germination (Mo- tissues, DMSO more readily diffuses ran et al, 1989a, b; Wickneswari and Nor- across cell membranes than other extrac- wati, 1991). tion buffer components, or selectively The loci and allozyme variants inferred transports certain buffer components, re- herein are considered putative as they are sulting in adverse internal concentrations not fully supported by segregation studies. detrimental on enzyme activity. Thus, However, consistent scoring was possible DMSO-treated tissues might show de- across the populations investigated with creased activity primarily for this reason the proposed genetic models. Some of and in addition to any species specific or these genetic models have been used suc- dilution effects. In contrast, with mechani- cessfully to study the mating system using cally homogenized extracts treated with single-tree progeny genotype arrays (Kha- sa et al, 1993b). Analysis of progeny ar- DMSO, cells are disrupted, and the protec- rays from controlled crosses or half-sib tive agents of the extraction buffer might families is necessary to confirm the genetic be able to reach and better protect the en- models, especially for loci where null al- zymes, likely resulting in a net positive ef- leles were detected. This is the case for fect of DMSO relative to tissues homoge- Est&num;1, Est&num;2, Est&num;3, Lap&num;1, Lap&num;2, and nized in buffer only. Further investigations Pgm&num;3. Among the loci scored, Moran et are needed to clarify these questions. As al (1989a, b) did not report null alleles. T was slightly superior to T for Racosper- 1 2 However, null alleles were reported in La- ma, it also seems to freeze better the Ra- rix laricina and other conifers (Cheliak and cosperma embryos without the extraction Pitel, 1985; lewandowski and Mejnartow- buffer. Probably, in presence of extraction icz, 1990), and in Terminalia superba buffer, the cell walls may rupture more (Vigneron, 1984). The presence of null al- quickly than its in absence, then exposing leles may be related to seed aging, either enzymes to denaturing secondary metabo- from prestorage treatment, or due to long lites. storage times in the seed bank (Cheliak Furthermore, for all operations, except and Pitel, 1985), or due to artifacts result- incubation, it is essential to keep the tem- ing in using systems with synthetic sub- perature at or below 4 °C. Even if an array (Pitel et al 1987). strates of operating temperatures was not tested Regarding enzyme subunit composition in this study, it has generally been demon- and compartmentalization, little information strated that when the temperature is above exists on tropical species (Vigneron, 1984; 40 °C, the activity of most enzymes de- Wickneswari, 1991).No such information severely (Pitel and Cheliak, creases exists in Racosperma studies by Moran et 1986a). Finally, the pretreatments to en- al (1989a, b). As suggested by Bousquet sure uniform germination are of great im- et a/ (1987) and Crawford (1990), the use samples analysed portance are as many of pollen as the source of enzymes and the differential germina- simultaneously and as examination of the progeny of seeds from tion of seeds could bias results of allozyme controlled crosses or half-sib families, variation in the population (Crawford, should be undertaken to study the active 1990). In fact, scarifying seeds with H 4 SO 2 subunit compositions of some enzymes instead of hot water gave better enzyme such as AAT, IDH, MDH, ME, which did activity especially for R auriculiforme while not clearly agree with the published litera- ture (Crawford, 1990; Kephart, 1990). hot water was better for R mangium. Nick-
JA Pitel for his valuable advice and L Clark for Modifying effects of enzyme subunit struc- her technical assistance during this work. This during extraction and/or electrophoret- ture work was supported by a grant form the Canadi- ic procedures may occur, producing atypi- an Intemational Development Agency (CIDA) to Such cal heterozygotes. atypical PD Khasa and a grant from the Fonds Québé- heterozygous isozyme patterns may result cois Pour la Formation des Checheurs et from both asymmetry of position or asym- Avancement de la Recherche (FCAR, ER-0693) to J Bousquet. Special thanks go to the CSIRO metry of staining intensity (up to total loss) (Australia), CTFT (Congo) and CTFK-SNR of one homopolymeric or the heteropoly- (Zaïre) for providing us with the plant material. meric form (Richardson et al, 1986). In view of the important variation in en- zyme activity and resolution of banding REFERENCES patterns encountered by using different tissue preparations, different electropho- WT, Birkes DS (1991) Estimating mating Adams resis buffer pH’s as well as the significant in forest tree populations. In: Bio- patterns variation between species and enzymes, chemical Markers in the Population Genetics it seems necessary, for every new spe- of Forest Trees (Fineschi S, Malvoti ME, cies being studied, to assess the effects Cannata F, Hattemer HH, eds) SPB Acad of extraction buffers, buffer systems, and Publ, The Hague, 157-172 conditions of material storage and han- Andrews A (1981) Electrophoresis: Theory, dling on the activity of a number of en- Techniques, and Biochemical and Clinical Applications. Clarendon Press, Oxford zyme systems, ideally in a factorial de- sign. Furthermore, a genetic base that is Bousquet J, Cheliak WM, Lalonde M (1987) Al- lozyme variability in natural populations of broad enough to be representative of allo- green alder (Alnus crispa) in Quebec. Ge- zyme variants likely to be encountered for 29, 345-352 nome the species of interest should be used. In Cheliak WM, Pitel JA (1984) Techniques for addition, within any good buffer system, Starch Gel Electrophoresis of Enzymes from this study showed that different pH condi- Forest Tree Species. For Canada, Petawawa tions of the buffer system may be re- Nat For Inst, Inf Rep PI-X-42 quired to obtain optimal enzyme resolu- Cheliak WM, Pitel JA (1985) Inheritance and tion and staining, all other sources of linkage of allozymes in Larix laricina. Silv variation being held constant. If accurate Genet 34, 142-148 analytical methods are developed at an Conkle MT, Hodgskiss PD, Nunnally LB, Hunter early stage, the number of loci and alleles SC (1982) Starch Gel Electrophoresis of will be maximized and the genetic infer- Conifer Seeds: A Laboratory Manual. USDA For Serv, Gen Tech Rep PSW-64 ence of allozyme variants will be more consistent and less subject to unexpected Crawford DJ (1990) Plant Molecular Systemat- ics: Macromolecular Approaches. John Wiley adjustments in the course of or after the and Sons, NY experimental phase of the study. YA (1991) Genetic variation within El-Kassaby and among conifer populations: Review and evaluation of methods. In: Biochemical Mark- ACKNOWLEDGMENTS ers in the Population Genetics of Forest Trees (Fineschi S, Malvoti ME, Cannata F, Hattemer HH, eds) SPB Academic Publ, The We grateful to TJB Boyle for allowing us to are Hague, 61-76 his laboratory facilities. We thank H Gurde- use ley, R Wickneswari, G Bellemare and C Trem- Gan YY, Robertson FW, Soepadmo E (1981) blay for suggesting improvements on earlier Isozyme variation in some rain forest trees. drafts of this manuscript. We also thank the late Biotropica 13, 20-28
Gelfi C, Righetti PG (1984) Swelling kinetics of Liengsiri C, Piewluang C, Boyle TJB (1990a) immobiline gels for isoelectric focusing. Elec- Starch Gel Electrophoresis of Tropical Trees: A Manual. ASEAN-Can For Tree Seed Cen- trophoresis 5, 257-262 tre, Muak Lek, Saraburi, Thailand Grodon M, Huang X, Peutonewy S, Zare R (1988) Capillary electrophoresis. Science Liengsiri C, Piewluang C, Boyle TJB (1990b) 242, 224-228 Characterization of isozymes of three tropical tree species: effect of extraction and running Postel W, Günther S, Weser J, Strahler Görg A, buffers on staining intensity and resolution. JR, Hanash SM, Somerlot L, Kuick R (1988a) J Trop For Sci 3, 111-122 Approach to stationary two-dimensional pat- tern: influence of focusing time and immobi- Loomis ND (1974) Overcoming problems of phe- line/carrier ampholytes concentrations. Elec- nolics and quinones in the isolation of plant trophoresis 9, 37-46 enzymes and organelles. Methods Enzymol 31, 528-544 Postel W, Günther S (1988b) The cur- Görg A, rent state of two-dimensional electrophoresis McLellan T (1984) Molecular charge and electro- with immobilized pH gradients. Electrophore- phoretic mobility in cetacean myoglobins of sis 9, 531-546 known sequence. Biochem Genet 22, 181- 200 Hamrick JL, Loveless MD (1986) Isozyme varia- tion in tropical trees: procedures and prelimi- McLellan T, Inouye LS (1986) The sensitivity of nary results. Biotropica 18, 201-207 isoelectric focusing and electrophoresis in the detection of sequence differences in pro- Hamrick JL, Loveless MD (1989) The genetic teins. Biochem Genet 24, 571-577 structure of tropical tree populations: associa- tions with reproductive biology. In: The Evolu- McLellan T, Sherman F (1991) The influence of tionary Ecology of Plants (Bock JH, Linhart YB, site-specificity of single amino acid substitu- eds) Westview Press, Boulder, CO, 129-146 tions on electrophoretic separation of yeast iso-1-cytochrome C. J Mol Evol 33, 395-401 Hartl DL, Clark AG (1989) Principles of Popula- tion Genetics. Sinauer, Sunderland, MA, 2nd McLellan T, Giovanna FLA, Nikaido K (1983) edn Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel Kephart SR (1990) Starch gel electrophoresis of electrophroesis. Genetics 104, 381-390 plant isozymes: a comparative analysis of techniques. Am J Bot 77, 693-712 Mitton JB (1983) Conifers. In: Isozymes in Plant Genetics and Breeding (Tanksley SD, Orton Khasa PD (1992) Scarification of Limba seeds TJ, eds) Elsevier, Amsterdam, part B, 443- with hot water, bleach, and acid. Trop Tree 472 Planters’Notes 43 (in press) GF, Muona O, Bell JC (1989a) Breeding Moran Khasa PD (1993) Acid scarification and hot wa- systems and genetic diversity in Acacia auri- ter soaking of Racosperma auriculiforme culiformis and A crassicarpa. Biotropica 21, seeds. For Chron (in press) 250-256 Khasa PD, Cheliak WM, Bousquet J (1993a) GF, Muona O, Bell JC (1989b) Acacia Moran Genetic variation in 26 populations of Racos- tropical forest tree of the coastal mangium: perma auriculiforme and R mangium using a genetic diversity. Evolution lowlands with low allozyme markers. Can J For Res (submitted) 43, 231-235 Khasa PD, Cheliak WM, Bousquet J (1993b) Nevo E (1978) Genetic variation in natural popu- Mating system of Racosperma auriculiforme lations: patterns and theory. Theor Pop Biol in a seed production area in Zaire. Can J Bot 13, 121-177 (submitted) Pitel JA, Cheliak WM (1984) Effect of Extraction Lewandowski A, Mejnartowicz L (1990) Inheri- Buffers on Characterization of Isoenzymes tance of allozymes in Larix decidua Mill. Silv from Vegetative Tissues of Five Conifer Spe- Genet 39, 184-188 cies: A User’s Manual. For Can Petawawa, (1991) Twenty-five years ago in Lewontin RC Nat For Inst, Inf Rep PI-X-34 genetics: electrophoresis in the develpment Pitel JA, Cheliak WM (1986a) Enzyme activities of evolutionary genetics: milestone or mill- during imbibition and germination of seeds of stone? Genetics 128, 657-662
Rhoades D, Cates R (1976) Toward a general tamarack Plant 67, (Larix laricina). Physiol theory of plant antiherbivore chemistry. Re- 562-569 cent Adv Phytochem 10, 168-213 Pitel JA, Cheliak WM (1986b) Effectiveness of Santi F, Lemoine M ( Genetic markers for ’390) for increasing activity of protective agents Prunus avium L: inheritance and linkage of five enzymes from vegetative tissues of isozyme loci. Ann Sci For 47, 131-139 white spruce. Can J Bot 64, 39-44 Variabilité génétique des prov- P (1984) Vignern Pitel JA, Cheliak WM (1988) Metabolism of en- Ivoriennes et Congolaises de Termi- enances zyme with imbibition and germination of nalia superba Engler et Diels: apports de pol- seeds of jack pine (Pinus banksiana). Can J Thèse de ymorphisme enzymatique. Bot 66, 542-547 doctorat, Univ Paris-Sud, France Pitel JA, Cheliak WM, Barreti J (1987) Inheri- Walters TW, Posluszny V, Kevan PG (1989) Iso- tance of allozymes in a black spruce diallel zyme analysis of the grape (Vitis). I. A practi- cross. Silv Genet 36, 149-153 cal solution. Can J Bot 67, 2894-2899 Pitel JA, Cheliak WM, Wang BSP (1989) Some Ackerman F (1983) A New Germi- Wang BSP, biochemical changes associated with stratifi- nation Box for Tree Seed Testing. For Can cation and germination of basswood seeds. Petawawa Nat For Inst, Inf Rep PI-X-27 Seed Sci Technol 17, 57-71 Wickneswari R (1991) Development of biochem- Powell JR (1983) Measuring genetic variation in ical genetic markers for tropical rainforest natural populations: where are we? In: Popu- species. In: Proc Malaysian Biochem Soc lation Biology: Retrospect and Prospect Conf 16, 6-15 (King CE, Dawson PS, eds) Columbia Univ Wickneswari R, Norwati M (1991) Techniques Press, NY, 97-115 for starch gel electrophoresis for enzymes Richardson BJ, Baverstock PR, Adams M from acacias. In: ACIAR Workshop on Hy- (1986) Allozyme Electrophoresis - A Hand- bridization and Vegetative Propagation of book for Animal Systematics and Population Australian Tropical Acacias. 1-4 July, Taw- Studies. Academic Press, Orlando, FL Sabah, Malysia au,