Respiration in a polyploid series in Saccharomyces

Respiration

in a Polyploid Maurice

From

the Biological

Series in Saccharomyces Ogur

Research Laboratory, Southern Carbondale, Illinois Received

May

Illinois

University,

13, 1954

INTRODUCTION Continuing interest in polyploidy is stimulated by its relationships to both the development of economically superior plants and to certain types of tumors. The construction of a polyploid series through tetraploid in Saccharomyces, validated by genetic (l), biochemical (2), and radiation (3) criteria, in the absence of adequate methods of direct chromosome count, has provided a model system for studying some of the metabolic effects of polyploidy in the yeasts. An earlier paper (2) reported that the average cellular content of deoxyribonucleic acid, ribonucleic acid, metaphosphate, and dry weight of haploid, diploid, triploid, and tetraploid Saccharomyces showed substantially integral dependence upon the number of chromosomal sets. Since the ratio of two integrally ploidy-dependent characters must be ploidy independent, a necessary corollary follows, namely, that any analytical quantity measured in this polyploid series found to be ploidy independent on a gravimetric basis must be ploidy dependent on a cellular basis. Some preliminary observations on the respiration of diploid strains and their parent haploids yielded rates of comparable magnitude when calculated to dry weight. It seemed of some interest, therefore, to study whether these rates were indeed ploidy dependent when cell counts were introduced. This report deals with oxygen uptake and carbon dioxide production in air of haploid, diploid, triploid, and tetraploid yeasts and will show that both increase with ploidy in approximately integral fashion when grown under comparable conditions and calculated to a cellular basis. 484

RESPIRATION

IN POLYPLOID

SACCHAROMYCES

485

EXPERIMENTAL

Yeast Cultures Haploid and diploid gametic cultures used in this study were from the Carbondale collection. These stocks, which have their origin in single ascospores obtained by microdissection of four spored asci, have been developed by a program of inbreeding and selection for various biochemical markers, and are nonsporulating. Stock cultures are stored on nutrient agar slants under mineral oil at 4°C. The nutrient agar medium employed for stock cultures or fresh transfers contained 4.0 ml. liquid yeast extract (Anheuser-Busch #3), 3.5 g. peptone, 0.5 g. MgSOl ,2.0 g. KHzPOl ,40 g. glucose, 30 g. agar, 206 pg. thiamine hydrochloride, 200 lg. calcium pantothenate, 200 pg. nicotinic acid, 200 pg. pyridoxine, 50 pg. paminobenzoic acid, and 10 pg. inositol per liter. A complete liquid medium employed differed from the above only by the omission of the agar and the reduction of the glucose concentration to 2%. Hybrid cultures were prepared by the Lindegren (4) mass-mating technique in either liquid media or on the surface of agar slants. The mating mixture was plated by spreading an appropriate dilution on the surface of a rotated nutrient agar plate, Hybrid colonies were selected after 48 hr. incubation at 30°C. by microscopic examination of cell size and shape and streaked on nutrient agar slants. Since all gametic stocks were nonsporulating, confirmation of successful hybridization could be obtained by sporulation. All hybrids included in this report formed spores when tested. One stored clone of haploid origin sporulated and was discarded. All haploids in this report failed to sporulate under the test conditions. The sporulation test medium contained 2.5 ml. of liquid yeast extract, 10 g. sodium acetate, and 30 g. agar per liter. Hybrids placed on acetate sporulation slants generally developed asci in 24-48 hr.

Methods Fresh 24-hr. slants were used as inocula for 250-ml. Erlenmeyer flasks containing 40 ml. of complete liquid medium. These flasks were shaken mechanically at 75 oscillations/min. with an amplitude of 3 in. for 18-24 hr. at 30°C. Cells were harvested by centrifugation at O-4%., washed three times with cold distilled water, and suspended in 50 ml. of distilled water. Dry weights were determined by weighing residues obtained by evaporating 20-ml. aliquots in tared aluminum pans at 70°C. in a forced draft oven for 6-8 hr. Appropriate dilution, generally 5 to 50, in 0.067 M (M/15)KH1POa (pH 4.6) was made to a turbidity represented by a scale reading in the vicinity of 100 in the Klett-Summerson photocolorimeter using the blue (420 rnp) filter. This yielded a suspension suitable for cell counts, total nitrogen assay, and respiration experiments. Cells were counted in a hemacytometer by conventional red cell procedures. Buds were scored as cells as in earlier work (2). In most of the cultures studied, particularly the polyploids, the growth conditions led to clones with very low percentage of buds. The treatment of the data would thus not depend greatly on the evaluation of the buds. The clustering growth habit of most haploid clones in-

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creases the uncertainty of scoring haploid cells. A number of haploid parents of hybrids included in the study had to be rejected as essentially uncountable owing to clumping. Total nitrogen was estimated photocolorimetrically by digestion and direct nesslerieation. Respiration and aerobic fermentation of yeast suspensions were measured by the direct method of Warburg with a shaking rate of 100 oscillations/min. at 30°C. All flasks contained 2.2 ml. of cells in the main compartment and 0.5 ml. of 3% glucose in M/15 KHzPO, in a side arm. Center wells contained 0.3 ml. of either 10% KOH or M/15 KHzPOI . Sugar was tipped in after 30 min. of endogenous observation, and respiration rates were calculated from regions of essentially linear slope, generally between 30 and 150 min. after glucose was tipped in. RESULTS

Yeasts Grown with Aeratim

The shaker growth conditions employed produced cultures which in most cases exhibited respiratory quotient (R.Q.) values cu. 2 with glucose as substrate, characteristic of baker’s yeast grown aerobically. The data for 15 haploids, 12 diploids, 8 triploids, and 1 tetraploid are summarized in Table I. Standard deviations of the individual clones from the mean for the ploidy category have been calculated in each case except the tetraploid where the mean and deviations are based on triplicate runs with a single representative of the category. The haploids showed the greatest individual differences in respiratory ability, as the magnitude of the deviations indicates. This does not appear to be related to the problem of haploid clusters, at least as far as cell scoring is concerned, since the deviations are large on the basis of dry weight or nitrogen as well as on a cellular basis. Since the haploids studitid origiTABLE Respiration

and Aerobic

Fermentation

in

I a Polyploid

Series

0% Q (cellp Haploid (15)d Diploid (12) Triploid (8) Tetraploid (1)

7.93 16.5 24.6 33.2

a Microliters/hr./cell b Microlitem/hr./mg. c Mioroliters/hr./mg. d Number of different * Standard deviation

zt f f f

2.7* 2.7 3.3 3.4

-

Q (Wb 792 1064 1148 1163

f f f zt

X 107. nitrogen. dry weight. clonea analyzed. = m.

284 86 95 118

Q (cell)

Q" 60.4 02.6 62.3 71.9

+ f zt f

19.4 7.5 6.6, 3.1

19.3 f 33.3 f 49.9 f 77.5 rt

4.2 6.7 6.3 14.2

in Saccharomyces CO* (air)

Q (N) 1936 2135 2225 2530

f 96 f 249 f 61 f 71

Q 134.5 128.6 121 158.5

f 27.5 f 23.5 * 9.7 f 8.5

RESPIRATION

IN

POLYPLOID

487

SACCHAROMYCES

nate in inbreds selected for biochemical deficiencies, some weaknesses in metabolic ability are to be anticipated. A few haploids of weak respiration which have been included in the aerobic haploid category contribute to this range. Aerobic-deficient haploids will be considered separately. The data in Table I indicate that oxygen uptake and carbon dioxide production in air calculated to dry weight or total nitrogen are essentially ploidy independent. Introduction of cell numbers, however, transforms the data into a ploidy series which shows integral dependence; diploid, triploid, and tetraploid values are essentially two, three and four times the haploid level. The smaller range observed for the diploid and triploid clones suggested that hybridization may often repair quantitative deficiencies in the respiratory mechanism in the parents. Aerobic-Sujicient Hybrids with One Aerobic-Dejicient Parent The aerobic-sufficient hybrids, which are obtained in most crosses between aerobic-deficient and aerobic-sufficient parental clones, afforded a convenient test system for evaluating the repair of respiratory deficiency in a cross, i.e., whether or not the integral ratio in cellular respiration in the polyploid series represented simple additivity of the parental abilities or was truly governed by the chromosomal complement. The data for two such crossesgiven in Table II indicate that the aerobic-sufficient hybrids formed in crosseswhere one parent was aerobic deficient exhibited their full ploidy-dependent respiration. Aerobic-Dejicient Yeasts Haploids with no measurable respiration under the test conditions showed enhanced carbon dioxide production in air compared to aerobicsufficient yeasts. Whereas a QG& (N) cu. 2000 was observed for the TABLE Respiration

of Aerobic-Suficient Culture 15469 8256 15469 x 8256-A 15467 11296 15467 X 11296-L

Hybrids

II with Ploidy

1 1 2 1 2 3

one Aerobic-Deficient

Parent Qor(cell)

cu. 0 9.04 18.1 ea. 0 13.6 27.3

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aerobic sufficients grown aerobically, a Qz’, (N) for 11 aerobic deficients cu. 3500 was observed after aerobic growth. This was comparable to the fermentation of aerobic-sufficient yeasts tested in a nitrogen atmosphere. Three commercial samples of baker’s yeast yielded a Q,“,2, (N) cu. 3500. Slonimski (5) has already reported an aerobic fermentation as strong as the anaerobic fermentation for the “petite colony mutant.” It was of interest, therefore, to compare the fermentation data for the aerobic-deficient and aerobic-sufficient yeasts on a cellular basis after growth with agitation. Whereas the aerobic-sufficient haploids yielded a QGE2(cell) cu. 19, a value cu. 30 was observed for the aerobic-deficient haploids. Yeasts Grown in Quiet Culture To gain some insight into the effect of cultural conditions on ploidydependent respiration and aerobic fermentation and into the special problem of the aerobic-deficient yeasts, a few experiments were carried out on yeasts grown without agitation. The effect of anaerobic growth in raising the QcO, and in decreasing the Q,,, has already been described (6). Our experiments confirmed these effects of partial anaerobiosis and, moreover, indicated that ploidy dependence was still exhibited on an altered numerical scale which depended on the growth conditions for the aerobic-sufficient yeasts. The enhanced CO2 production in air of aerobic-sufficient yeasts grown in quiet culture was comparable to that of aerobic-deficient yeasts grown in quiet culture or with aeration. DISCUSSION

Published studies concerning the genetic basis of enzyme activity have been primarily concerned with the qualitative demonstration of the presence of the enzyme in the dominant allele and its absence at levels which can be measured in the recessive. Relatively little information appears to be available concerning the quantitative aspects of enzymatic activity as a function of gene dosageas in heterozygous and homozygous hybrids or as a function of the total chromosomal complement as in polyploidy. The lack of polyploid systems consisting of discrete cells which may be scored readily has undoubtedly delayed such studies. Although haploid and diploid yeasts have been recognized for at least 15 years, dependence of respiration upon the number of chromosomal sets does not appear to have been observed. The prevailing tendency to express respiration data in terms of dry weight or nitrogen assays (which

RESPIRATION

IN

POLYPLOID

SACCHAROMYCES

489

are themselves ploidy dependent in Saccharomyces on a cellular basis) rather than in terms of cell numbers yields comparable rates for most haploids and diploids. The construction of a polyploid series of aerobic-sufficient yeasts and the observation that cellular dry weight is itself ploidy dependent (2) made possible the current demonstration of an increase in cellular respiration and aerobic fermentation proportionate to the chromosomal complement. These findings are interpreted as representing control of the quantitative enzyme potential by the quantitative genetic complement of the cell. Whether such control is exercised as the direct expression of the gene-enzyme relationship or as the indirect expression of nuclear control of cell size and size-related characteristics is not yet apparent. A number of other variables affecting the respiration and fermentation of yeasts superimposed on the ploidy-dependent variable are already appreciated. Baker’s yeast itself represents a genetic selection for strong fermentative power with a relatively limited aerobic capacity, metabolizing carbohydrate almost exclusively via aerobic pathways at low substrate levels but showing considerable aerobic fermentation at high substrate levels. Further selection for respiratory capacity is to be expected in any extensive program of inbreeding haploid yeasts for nutritional deficiencies. The greater range of respiratory ability in the haploid category of the current study may be so interpreted. Hybrids, heterozygous for a deficiency, which exhibit the qualitative characteristics of the dominant allele, are of course normally expected. The realization of the full quantitative ploidy-dependent aerobic potential in the cases of hybrids formed by crossing aerobic-deficient by aerobic-sufficient parents rather than simply that of the sufficient parent or an intermediate value between the parental values is consistent with the interpretation that repair of a deficiency, presumably extrachromosomal, will lead to the reassertion of genetic control of quantitative enzyme potential. The environmental variation of genetically controlled phenotypes must also be considered. Dependence of the respiratory and fermentative mechanisms in the yeasts on the degree of aeration during growth has been recognized previously (6) and observed in the current work. For purposes of ploidy-dependence study, comparable conditions of growth are, therefore, required. Such comparable conditions are more difficult to achieve between the extremes of vigorous aeration and total anaerobiosis. Vigorous aeration of aerobic-sufficient and aerobic-deficient yeasts

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does not render the growth conditions comparable. Cellular levels of fermentation of aerobic-deficient yeasts may be compared with aerobicsufficient yeasts grown in quiet culture. ACKNOWLEDGMENTS This work was supported in part by grants from the U. S. Public Health Service and Anheuser-Busch, Inc. The author wishes to thank Dr. Carl C. Lindegren and Mrs. Gertrude Lindegren for helpful discussions. Mating cultures were from the Carbondale stocks. Some of the hybrids used were prepared by Mr. Jaun de Dios Calle, by Mr. Alvin Sarschek, and by Mrs. Gertrude Lindegren. The acetate agar sporulation medium was suggested by Mr. Alvin Sarachek. SUMMARY

1. A polyploid series from haploid through tetraploid in Saccharomyces has been analyzed for respiration and aerobic fermentation with glucose as substrate. 2. Q,,, (cell) and Q$b’, (cell) increase in integral fashion with ploidy in cultures grown under comparable conditions. 3. Respiration-sufficient hybrids with one respiration-deficient parent exhibit the full ploidy-dependent respiration of the hybrid. 4. The findings are interpreted as dependence of quantitative enzyme potential on the quantitative genetic complement. REFERENCES 1. LINDEGREN, 2. OGUR, M.,

C. C., AND LINDEGREN, G., J. Gen. Microbial. 6, 885 (1951). MINCKLER, S., LINDEGREN, G., AND LINDEGREN, C. C., Arch. Biothem. and Biophys. 40, 175 (1952). 3. LUCRE, W. H., AND SARACHEK, A., Nature 171, 1014 (1953). 4. LINDEGREN, C. C., AND LINDEQREN, G., Proc. Natl. Acad. Sci. U. S. 29, 306 (1943). 5. SLONIMSKI, 6. EPHRUSSI,

P. P., Ann. inst. Pasteur 76, 510 (1949). B., AND SLONIMSKI, P. P., Biochem. et Biophys.

Acta

6, 256 (1950).