(1982) 70, 1710-1714. 0032-0889/82/70/17 10/05/$00.50/0. Ploidy Effects in Isogenic Populations of Alfalfa'. II. PHOTOSYNTHESIS, CHLOROPLAST NUMBER ...
Plant Physiol. (1982) 70, 1710-1714 0032-0889/82/70/17 10/05/$00.50/0
Ploidy Effects in Isogenic Populations of Alfalfa' II. PHOTOSYNTHESIS, CHLOROPLAST NUMBER, RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE, CHLOROPHYLL, AND DNA IN PROTOPLASTS Received for publication May 13, 1982 and in revised form August 6, 1982
WILLIAM T. MOLIN, STEVEN P. MEYERS, GIANNI R. BAER, AND LARRY E. SCHRADER2 Department ofAgronomy, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT Photosynthetically-active protoplasts isolated from isogenic sets of dip-
loid-tetraploid and tetraplold-octoplold alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5bisphosphate carboxylase (RuBPCase), chlorophyll (Chi), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number ofchloroplasts and amount of ChW per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploWd population. The rate of C02dependent 02 evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraplold to octoploid level. Whereas leaves and-protoplasts had simflar increases in RuBPCase, DNA, and Chi with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.
A better understanding of the physiological consequences of polyploidization should facilitate the development of agronomically useful genotypes from experimental polyploids. A principal difficulty inherent in physiological comparisons of polyploids is that cell volume (18), chloroplast number per cell (2), and the amount of DNA per nucleus (5) have been shown, in some cases, not to increase in exact proportion to an increase in genome size. In these instances, data expressed on a fresh weight, leaf area, or Chl basis may be misleading if the increase in these characteristics in relation to ploidy level has not been determined. The preferred method of comparing polyploids is to isolate protoplasts so that the amount of DNA can be correlated with genome size, and physiological data can be expressed on a per cell basis. Protoplast isolation also eliminates variability resulting from differences such as stomatal density (14) or anatomical characteristics (3). Allopolyploids (3, 7, 12) and colchicine-induced polyploids (14, 18) have been used to study the effects of polyploidization on biochemical and physiological processes. Results from these studies are confounded by genetic differences in the case of allopolyploids and problems associated with inbreeding depression in colchicine-induced and spontaneously appearing polyploids. Au-
topolyploids of alfalfa (11) avoid these influences and allow a more direct study of polyploidization. These polyploids consist of an isogenic diploid-tetraploid (DDC 2X-4X) population of alfalfa to which maximum heterozygosity was restored and an isogenic tetraploid-octoploid (IC 4X-8X) population that was passed through a single sexual cycle, thus restoring some heterozygosity to the plants. These isogenic alfalfa populations differ only in the level of ploidy and can be used to ascertain the effects of polyploidization on physiological and biochemical processes. We have recently developed techniques for alfalfa protoplast isolation and a sensitive fluorometric technique for DNA quantitation (1). These techniques have permitted us to examine the consequences of polyploidization at cellular and chloroplast levels and to express data on a per cell basis.
MATERIALS AND METHODS
Plant Material and Culture. Alfalfa (Medicago sativa L.) plants were grown in a growth chamber as described by Meyers et al. (9). Protoplast Isolation. Fully expanded trifoliolate leaves (from the third to sixth node from the apex on plants 4 weeks after cutting) were harvested for protoplast isolation. A minimum of five replicates (pots) were used for each experiment with two plants per pot comprising a replicate. The leaflets were washed in distilled H20 and sliced into 1-mm sections. Tissue (0.7 g) was placed in 25 ml of the following medium: 0.6 M sorbitol, 5 mm Mes (pH 5.6), 2 mm CaCl2, 0.25% (w/v) BSA, 0.1% (w/v) Pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan), 1.0%/ (w/v) Rhozyme HP150 (Rohm and Haas), and 1.0%0o (w/v) Onosuka cellulase (Yakult Honsha Co., Tokyo, Japan). The rhozyme and cellulase were desalted by a single passage through a column (25 x 110 mm) of Biogel-P6 (Bio-Rad) in 10 mm citrate buffer (pH 5.0) prior to use. The suspension was incubated for 3 h at 30°C. Large, undigested leaf fragments were separated from the protoplasts by filtrations through nylon cloth (80 ,um mesh, Tetko Inc.,
Elmsford, NY). The protoplasts were collected by centrifugation at lOOg for 10 min. Subsequent procedures were performed at 4°C. The supernatant was discarded and the protoplasts were
suspended in 40 ml of 0.6 M sucrose, 5 mM Mes (pH 6.0), 2 mM CaCl2, and 12% (w/v) dextran (40,000 mol wt, Sigma) by gently swirling the tubes. Ten ml of protoplast suspension were placed in each of four 10-ml volumetric flasks. The protoplast suspension was overlaid with 0.5 ml of 0.6 M sorbitol, 5 mm Mes (pH 6.0), 2 mM CaCl2, and 0.05% (w/v) BSA. The flasks were centrifuged at 400g for 10 min during which time the protoplasts floated to the sorbitol/sucrose interface. The protoplasts were gently removed with a Pasteur pipette, suspended in 10 volumes of 0.6 M sorbitol, 5 mM Mes (pH 6.0), 0.05% (w/v) BSA, and 2 mm CaCl2, and ' Supported by College of Agricultural and Life Sciences, University of pelleted by centrifugation at 100g for 10 min. For photosynthetic Wisconsin-Madison, and by United States Department of Agriculture studies, protoplast pellets were suspended in 0.6 M sorbitol, 50 mM Hepes (pH 7.6), 2 mm CaCl2, 0.05% (w/v) BSA, and 10 mM Competitive Research Grant 5901-0410-9-0361-0. NaHCO3 (buffer A). 'To whom correspondence should be addressed. 1710
PLOIDY EFFECTS IN ISOGENIC ALFALFA
Washing leaves prior to the isolation of protoplasts was an essential step in the procedure. The washing step was thought to remove insecticides, used to prevent spider mite infestations, from the leaf surface. Omission of this step resulted in protoplast preparations which would not float during the isolation procedure. The same result was obtained if the cellulytic enzymes were not desalted before use. Chloroplast Isolation. Protoplasts were suspended in 0.5 M sorbitol, 50 mm Hepes (pH 7.6), 2 mm EDTA, I mM MgCI2, I mM MnCl2, 10 mM NaHCO3, 0.2 mM NaH2PO4, and I mm Na2H2P207 (buffer B). The suspension was passed three times rapidly through 20-,gm mesh nylon cloth that had been attached to the end of a 1ml syringe. Chloroplasts from ruptured protoplasts were pelleted by centrifugation at 500g, and washed twice in buffer B. The pellet was resuspended in buffer B and stored on ice. The percentage intactness of chloroplasts was determined by the ferricyanide-dependent 02 evolution before and after osmotic shock according to Lilley et al. (8). By this criterion, chloroplasts isolated from alfalfa protoplasts were 89% intact. DNA, RuBPCase3, Chl, and C02-Dependent 02 Evolution. DNA in protoplasts was determined according to Baer et al. (1). RuBPCase content in protoplasts was determined by rocket immunoelectrophoresis using rabbit IgG raised against purified alfalfa RuBPCase (10). RuBPCase from protoplasts was solubilized by freezing and thawing a suspension twice and then sonicating the suspension for 10 s. Chl and the Chl a to b ratio were determined according to the procedure of Wintermans and DeMots (19) in 96% ethanol. C02-dependent O2 evolution was measured polarographically at 25°C using a Rank Brothers (Cambridge, England) electrode system. The assays contained 5 to 25 ,tg Chl in a volume of 2.0 ml. Light was provided by flood lamps giving a quantum flux density of 90 ,umol quanta m- s-. To prevent breakage of protoplasts and chloroplasts, the magnetic stirrer was set at a minimum speed while still maintaining a stable response. The assay contained 1.85 ml of deoxygenated buffer (buffer A for protoplasts and buffer B for chloroplasts), 0.05 ml of buffer A or B containing 400 mm NaHCO3, and 0.1 ml of protoplast or chloroplast suspension. The 0.05 ml of 400 mm NaHCO3 provided additional bicarbonate which may have been lost during deoxygenation. Chloroplast Counts and Cell Number. The cell number per ml was determined with a hemacytometer and was the average from counts of 12 fields with five 4-,ul volumes being counted per field. The number of chloroplasts per cell was determined according to the procedure of Cattolico (4). Five ,ul of protoplast suspension in photosynthesis buffer containing approximately 1 x 106 cells/ml was used to prepare chloroplasts for counting. It was assumed that each observation (count of one cell) was independent. Statistical Analysis. Independent sample t tests were performed to test the hypothesis of equal means for isogenic sets. A Kolmogorov-Smirnov test (16) was used because the distribution of the chloroplast per cell data was uncertain. Leaf Area Determinations. Leaf areas of trifoliolate leaves were determined on five replicates from one experiment with a Li-Cor LI-3 100 area meter. RESULTS Isolation of Protoplasts and Chloroplasts. The procedure described herein permitted the isolation of large quantities of intact, photosynthetically active protoplasts from alfalfa leaves and considerably reduced the time required for isolation compared to previously reported methods (6). The method was also effective in promoting the rapid release of protoplasts from expanding soy3Abbreviations: RuBPCase, ribulose- l,5-bisphosphate carboxylase; IgG, gamma immunoglobulin.
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bean (Glycine max [L.] Merr.) and Phaseolus leaves, and may have general application for the isolation of protoplasts from other legumes. Rates of C02-dependent 02 evolution increased 2- to 4fold when BSA (0.25% w/v) was included in the isolation medium (data not shown). The optimum concentration of osmoticum was found to be 0.6 M sorbitol. Centrifugation of protoplast suspensions in excess of lOOg caused rupture of protoplasts. Chloroplasts were easily obtained from protoplast preparations by passage through a 20-tum nylon cloth. Alfalfa chloroplasts were osmotically stable at 0.5 M sorbitol as determined by the ferricyanide-dependent O2 evolution assay (8). Comparison of Protoplasts. Protoplasts obtained from DDC 4X alfalfa contained twice as much DNA, Chi, RuBPCase, and nearly twice as many chloroplasts per cell as did the DDC 2X alfalfa (Table I). The rate of C02-dependent 02 evolution increased 1.93fold with a doubling in ploidy level (Table I). Ratios of Chl a to b, Chl to DNA, RuBPCase to DNA, and RuBPCase to Chl remained essentially constant with an increase in ploidy level from 2X to 4X (Table II). Protoplasts isolated from the IC 4X-8X set of alfalfa did not show a doubling with ploidy in characteristics as did the DDC 2X-4X set of alfalfa. The number of chloroplasts (Table I) and Table I. Effect of Polyploidization on DNA, RuBPCase, Chl, and 02 Evolution in Diploid-Tetraploid and Tetraploid-Octoploid Protoplasts Isolatedfrom Fully Expanded Trnfoliolate Leaves Ploidy Level DDC 2X DDC 4X IC 4X DNA (pg cell-') 6.4 12.4**a 12.5 RuBPCase (pg cell-') 128.6 247.3** 255.3 Chl (pg cell-l) 23.0 40.6** 44.2 4.2 8.1* 8.3 02 evolution (pmol 2 h-' cell-') Chloroplasts (no. cell-l) 21.4 40.2** 31.2 a * **denote significance at the 0.05 and 0.01 levels within genotypes of a pair, respectively.
IC 8X 17.2** 348.0** 80.8** 13.9
59.1** isogenic
Table II. Effect of Polyploidization on the Relationship between DNA, RuBPCase, Chl, 02 Evolution, and Chl a and b in Diploid-Tetraploid and Tetraploid-Octoploid Protoplasts Isolatedfrom Fully Expanded Trifoliolate Leaves Chl: DNA, RuBPCase:DNA, and RuBPCase:Chl ratios were calculated from data in Table I, and were not analyzed statistically. Ploidy Level
Ratio Chl:DNA RuBPCase:DNA RuBPCase:Chl Chl a:b 02 evolution (,mol 02 mg-' Chl h-')
DDC 2X DDC 4X IC 4X IC 8X 3.6 3.2 3.5 4.7 20.1 19.9 20.4 20.2 5.6 6.1 5.8 4.3 3.3 3.4 3.2 3.3 182.6 199.5 187.7 173.2
Table III. Effect of Polyploidization on Chloroplasts Obtainedfrom Diploid-Tetraploid and Tetraploid-Octoploid Protoplasts RuBPCase and Chl per chloroplast were calculated from data in Table I, and were not analyzed statistically. Ploidy Level pg RuBPCase chloroplast-' pg Chl chloroplast-' 02 evolution (umol 02 mg' Chl h-')
DDC 2X DDC 4X IC 4X 6.0 6.1 8.2 1.10 1.02 1.43 151.2 121.1 134.0
IC 8X 5.9 1.37 177.7
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MOLIN ET AL. Table IV. Analysis of Chloroplast Distribution per Cell
Ploidy DDC2X DDC4X IC4X IC8X
na 360 391 372 488
No. Chloroplast SE 21.4 ± 0.3 40.2 ± 0.5 31.2 ± 0.4 59.1 ± 0.6
V Vaine 26.675 84.641 60.628 195.965
Coefficient
of Skewness
4.46 6.36 3.94 6.34
0.76 1.02 0.93 0.93
Kolmogorov-Smimov
Kurtosis
D:Normal 0.075 0.075 0.093 0.062
value" PpVle
