Plant Physiol. (1976) 57, 812-816

Effect of Thiamine

on

Ethanol and Pyruvate Production in

Helminthosporium maydis1' 2 Received for publication November 26, 1975 and in revised form February 20, 1976

ROBERT C. EVANS3 Department of Botany, The Ohio State University, Columbus, Ohio 43210 MICHAEL 0. GARRAWAY Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210 and The Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 MATERIALS AND METHODS These experiments were carried out using a single spore isoGrowth of the fungus Helminthosporium maydis race T in a basal glucose-L-asparagine liquid medium, pH 5, is inhibited by thiamine-HCI. late of Helminthosporium maydis Nisikado and Miyake, race T. Analysis of the media for organic acids reveals that the extracegular This isolate was obtained from an ear of corn in a field in pyruvate concentration decreases as the thiamine-HCI concentration of Franklin County, Ohio in the summer of 1970. Its pathogenic the medium increases. Extracellular ethanol, in contrast to pyruvate, characteristics appear unchanged after 5 years in culture. Maintenance of Fungal Cultures. The viability of the fungus increases in concentration as the thiamine-HCI concentration of the was maintained by repeated transfers to a sequence of solid medium increases under both aerobic and anaerobic conditions. The changes in ethanol and pyruvate levels in the presence of thia- media. The fungus was grown first on a 2% (w/v) potatomine-HCI occur via a thiamine-mediated increase in the activity of dextrose-agar medium and then was transferred to a more depyruvate decarboxylase but not alcohol dehydrogenase. This increase in fined medium containing 10 g D-glucose, 2 g D-xylose, 4 g Lpyruvate decarboxylase activity appears to be due to an increase in the asparagine, 1.5 g KH2PO4, 0.75 g MgSO4-7 H20, and 20 g quantity of enzyme present rather than an activation of pre-existing Difco Bacto-agar/l distilled H20. The pH of this medium, hereafter denoted as glucose-xylose medium, was adjusted to 6 enzyme. Whereas thiamine-pyrophosphate stimulates pyruvate decarboxylase activity in vitro, thiamine-HCI has no effect. Neither thiamine before autoclaving at 15 p.s.i. for 15 min. Lastly, cores of tissue derivative affects alcohol dehydrogenase activity. The increase in pyru- from this medium were transferred to a third solid medium which differed from glucose-xylose medium only in that xylose vate decarboxylase activity which accompanies an increase in the thinmine-HCI concentration of the medium is correlated with a decrease in was omitted. This final solid medium will be denoted as glucose medium. This tissue maintenance protocol was restarted by the level of intracellular pyruvate. transferring cores of tissue from glucose medium back to potatodextrose-agar. All tissue grown on solid media was incubated in the dark at 28 C. Experimental Media. For all experiments, an attempt was made to ensure as uniform an inoculum source as possible. Eight 12-mm cores of tissue from 10-day-old cultures grown on gluRecently, Garraway (7) reported that thiamine can adversely cose medium were transferred to 50 ml of sterile liquid medium affect fungal reproduction by showing that sporulation by Hel- in cotton-stoppered 250-ml Erlenmeyer flasks. This basal meminthosporium maydis, the Southern Corn Leaf Blight patho- dium was similar to the solid glucose medium, except that no agar was added, and trace elements consisting of CuCl2, MnCl2, gen, is inhibited by over 70% when grown on a solid glucose-Lasparagine medium containing 1.0 ,g/ml thiamine. Garraway's ZnCl2, Fe2(SO4)3, and NaMoO4 were added to give a final study suggests that H. maydis might serve as a model system for concentration of 0.1 mg of each trace element/l of solution. Glucose and each of the trace elements were autoclaved sepaan examination of the physiological and biochemical events associated with a morphogenetic process such as sporulation. As rately. The nitrogen source, MgSO4, and KH2PO4 were autoclaved together, and all constituents were combined using asepa preliminary step to a more detailed study of sporulation proctic techniques. The pH of the nitrogen-sulfate-phosphate soluesses per se, we are investigating some of the ways in which thiamine affects the levels of certain metabolites in H. maydis. A tion was adjusted to 6 prior to autoclaving; after the addition of preliminary report has been published (5). Information gathered all constituents, the final pH was still 6. The cores in this medium were incubated on a shaker oscillatin this study has provided directions for a future examination of ing at 100 cycles/min at 26 C in the light for 2 days. At the end thiamine inhibition of sporulation. of this time, the spores and associated hyphae emanating from these spores were separated from the agar cores by decanting 1 This research was supported in part by a United States Department into an empty, presterilized 250-ml flask. Then, 0.5-ml aliquots of Agriculture Cooperative State Research Service grant, Contract No. of this hyphal material were dispensed into 50 ml of experimen215-15-38, to The Ohio Agricultural Research and Development Cental media using a sterile 1 0-ml pipette. In cases where the ter. amount of tissue was meager, the amount of tissue in each 2 A portion of this report is taken from a dissertation submitted by aliquot was increased by centrifuging the sample at low speed R.C.E. in partial fulfillment of the requirements for the Ph.D. degree in and dispensing 0.5-ml aliquots of the resuspended "pellet." Botany at The Ohio State University. The experimental media were identical to the liquid preincuCam3 Present address: Department of Biology, Rutgers University, bation medium described above except that the pH of the nitroden, N.J. 08102. 812 ABSTRACT

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Physiol. Vol. 57,

1976

THIAMINE EFFECTS IN HELMINTHOSPORIUM

gen-sulfate-phosphate solution was adjusted prior to autoclaving with 5 N HCl so as to give a final pH of 5 to the complete media and that thiamine-HCl was added to some of the flasks to give a final concentration of either 0.1 or 1.0 ,ug/ml. Harvesting and Sampling Procedures. The tissue was incubated for 3 days on shakers at 100 cycles/min at 26 C. At the end of this period, the tissue was harvested by straining through cheesecloth. The pH of the media was measured, and a 0.5-ml aliquot was placed in a 2-dram screw-cap vial containing 0.03 ml of concentrated HCl; each vial was stored at 4 C for later ethanol analysis. The remainder of the media was frozen and later assayed for pyruvate content. The tissue was washed with double-distilled H20 and placed in cotton-stoppered 250-ml Erlenmeyer flasks containing 50 ml of fresh media of the same composition as for the 3-day incubation. These flasks containing 3-day-old tissue in fresh media were replaced on the shaker and incubated for 6 hr. A 0.5-ml aliquot of the media from each flask was removed at 3 hr and 6 hr and stored as described before for ethanol analysis. At the end of 6 hr, the tissue was harvested, washed, and heated at 80 C for 24 hr for dry weight determination. The pH of the media was determined and the media frozen for later determination of pyruvate. In some cases, certain 3-day-old cultures were divided in half after washing and each half placed in 50 ml of fresh media in 250-ml suction flasks equipped with serum stoppers. One flask was flushed with N2 for 10 min and the other left open to the air. Then, both flasks were rubber-stoppered and incubated for 6 hr on the shaker. Media samples at 3 and 6 hr were taken by inserting a 1-ml syringe through the serum stopper and withdrawing the 0.5-ml aliquot. These samples were stored as described above and the dry weights were determined. Ethanol Analysis. The ethanol content of the media was analyzed using a Hewlett-Packard gas chromatograph Model 5751 B equipped with a dual column and a dual flame detector. The solid support was Chromosorb W-AW, 60-80 mesh; the liquid phase was 5% Epon 1001. The injection port temperature was 210 C, the flame detector 235 C, and oven 110 C. The identification of ethanol in the media was confirmed by cochromatography with commercial ethanol on three different columns and by fractional distillation of the media and subsequent gas chromatographic analysis of the fraction collected between 78 and 82 C.

Pyruvate Colorimetric Determination. Pyruvate in the culture medium was estimated by a modification of the method of Straub as outlined in Neish (13). In this method, 1 ml of a 2% (v/v) solution of salicylaldehyde in 95% ethanol and 0.5 ml of a mixture of 100 g KOH and 60 ml distilled H20 were added to 2 ml of media and heated for 10 min at 37 C, cooled for 10 min at room temperature, and the absorbance read at 470 nm. Addition of 1 ml of 20% (w/v) trichloroacetic acid to the medium before the addition of the salicylaldehyde and KOH resulted in no detectable protein which might interfere with the assay and thus that step was omitted from Straub's method. Pyruvate Determination by Paper Chromatography. Each type of culture medium was concentrated to 5 ml using a Buchler flash evaporator. A 10-,ul aliquot of the concentrate was spotted on Whatman No. 1 paper along with known a-keto acids and run in a chamber using 1-butanol-formic acid (95:5) (water-saturated) solvent after Magasanik and Umbarger (12). After running the paper in the solvent for 20 hr, the paper was air-dried and developed by spraying with a mixture of 0.1% (w/v) semicarbazide and 0.15% (w/v) sodium acetate (12). After heating at 110 C for 10 min, the semicarbazone spots were visualized under UV light. For quantitative analysis, the dried spots were cut from the paper and each placed in 3 ml of 0.025 % (w/v) 2,4dinitrophenylhydrazine in 0.5 N HCl for 10 min. Then 1 ml of 40% (w/v) KOH was added, and the mixture was shaken for 10 min

on a

rotary

shaker at 100

oscillations/min

to elute the

813

hydrazone. The hydrazone was then measured colorimetrically at 435 nm using a Spectronic-20 spectrophotometer (12). Pyruvate Decarboxylase Tissue Analysis. The assay for this enzyme utilized a modification of the procedure of Ullrich (18) and that of Witt and Heilmeyer (21). Frozen fungal tissue was macerated with a razor blade and then ground with sand in 10 ml of 0.01 M K-phosphate buffer, pH 7.6. The resulting brei was centrifuged at 10,000 rpm for 5 min at 4 C. The residue was discarded, and the supernatant was used in the enzyme assay. This supernatant was kept at 0 C until ready for use. The reaction mixture contained 3.3 mM MgSO4* 7 H20, 70 mM Na-succinate buffer (pH 6), 0.08 mm NADH, 12.2 mM sodium pyruvate, 7.5 mm thiamine pyrophosphate, 0.5 ml of a 1 mg/ml solution of commercial alcohol dehydrogenase in 0.01 M phosphate buffer (pH 7.6), and 0.2 ml of sample supematant. The total volume of the reaction mixture was 3 ml. The commercial alcohol dehydrogenase (from yeast) was obtained from the Sigma Chemical Co. and contained 200 mg (NH4)2SO4, 30 mg Na4P207, and 10 mg glycine/100 mg protein. The reaction was started by the addition of sample supernatant. The protein content of the sample supernatant was determined by a modification of the procedure of Lowry et al. (11) with BSA as the standard. One ml of 20% (w/v) trichloroacetic acid was added to 1 ml of sample supernatant. This solution was then centrifuged at 10,000 rpm for 5 min at 4 C. The protein pellet was resuspended in 1 ml of double-distilled H2O. To 0.5 ml of this aqueous protein solution was added 5 ml of cupric tartrate solution containing 4 g NaOH, 0.4 g sodium tartrate, 30 g Na2CO3 and 0.2 g CuSO4 5 H2O (first dissolved in 50 ml H20) in a total volume of 1 liter. After the addition of the cupric tartrate solution and briefly mixing, 0.5 ml of a 1:1 dilution of 1.2 N HCI and Folin-phenol reagent was added and mixed with the rest of the constituents. After 20 min, the absorbance was read at 625 nm on the Spectronic-20. Intracellular Pyruvate Analysis. Intracellular pyruvate was measured enzymically using a modification of the procedure of Kubowitz and Ott as outlined in Kornberg (10). The reaction involved measuring the change in absorbance as NADH is converted to NAD in the reduction of pyruvate to lactic acid by lactic acid dehydrogenase (EC 1.1.1.27). The reaction mixture contained 0.08 mm NADH, 0.01 mm K-phosphate buffer (pH 7.6), 0.2 ug of commercial lactic dehydrogenase, and 0.2 ml of sample supernatant obtained as described for the pyruvate decarboxylase assay. All constituents were kept at 0 C immediately before use, and the reaction was started by the addition of sample supernatant. The lactic dehydrogenase was obtained from Sigma and was a crystalline suspension of rabbit muscle lactic dehydrogenase in 2.1 M (NH4)2SO4. The pyruvate was estimated by measuring the change in absorbance/min at 340 nm. Alcohol Dehydrogenase. Alcohol dehydrogenase was assayed using a modification of the method of Vallee and Hoch (19). Because H. maydis alcohol dehydrogenase was found to be inactivated by the grinding procedure outlined for pyruvate decarboxylase, an alternative protocol was used. Four frozen cultures of H. maydis tissue (approximately 4 g) were macerated with a razor blade and homogenized with 50 ml of ice-cold acetone in a VirTis homogenizer for 2 min at high speed. This brei was suction-filtered, and the residue was washed with additional cold acetone and allowed to air dry overnight. Then, 200 mg of this acetone powder was dissolved in 10 ml of ice-cold Kphosphate, pH 7.6, and centrifuged at 10,000 rpm for 5 min at 4 C. The supernatant was used for the enzyme assay; the residue was found to have negligible alcohol dehydrogenase activity. For the assay itself, the reaction mixture contained 0.01 mm K-phosphate (pH 7.6), 0.08 mm NADH, 6.5 mm acetaldehyde, and 0.3 ml of sample supernatant. All components were kept at

EVANS AND GARRAWAY

814

0 C until ready for use, and the reaction was started by the addition of sample supernatant. The enzyme was assayed by measuring the change in absorbance/min at 340 nm. The rate was expressed on an mg total protein basis using a modification of the method of Lowry et al. (11) as described previously. This same assay was used in an attempt to assay intracellular ethanol as well by substituting commercial alcohol dehydrogenase for the fungal enzyme and substituting 2.4 Amol NADI for the NADH. Even when 1 ml of sample supernatant (prepared as for the pyruvate assay) was used there was no detectable intracellular ethanol. Analysis of Data. All experiments were performed at least twice with at least three replicates per experiment. The results appearing in tables and figures are expressed as the mean of all replicates + SE.

RESULTS AND DISCUSSION The growth curve for H. maydis in liquid culture appears sigmoidal in shape (Fig. 1), which is typical of filamentous fungi. For cultures incubated in excess of 2 days there is less tissue produced when thiamine is present than when it is absent. No such inhibitory effect of thiamine on dry weight is seen for cultures incubated for 2 days or less. The deceleration phase of growth occurs approximately 1 day earlier when thiamine is present, and there appears to be no significant difference in dry weight between cultures grown in 0.1 versus 1.0 ,g/ml thiamine. Measurements of the pH of the culture media after 3 days of incubation reveal that there is a tendency for the pH to increase as the thiamine concentration of the media increases. This increase in pH in the presence of thiamine is observed regardless of the initial pH of the media and suggests that thiamine reduces the production and/or excretion of acids by the fungus. A preliminary thin layer chromatographic analysis of organic acids present in the culture media after 3 days of incubation demonstrated the presence of several different acids. However, only the a-ketoacids exhibited a concentration change in response to the addition of thiamine to the medium; and thus these acids were studied in more detail. An assay of a-ketoacids present in the media using paper chromatography indicated that the concentration of pyruvate was particularly sensitive to the thiamine concentration; the pyruvate content of the media decreased from 2.4 + 0.3 mg pyruvate/g dry weight when thiamine is absent to 1.5 ± 0.3 mg/g in the presence of 0.1 ,ug/ml thiamine and 0.8 ± 0.1 mg/g in the presence of 1.0 ,ug/ml

Plant

Physiol. Vol. 57,

1976

thiamine. The concentrations of a-ketoglutarate and oxaloacetate in the media appeared to decrease much less than pyruvate does in the presence of increasing thiamine concentrations. Two locations where pyruvate and thiamine are often closely associated occur at the end of glycolysis. Because of the requirement for thiamine by pyruvate decarboxylase and pyruvate dehydrogenase in many organisms, it was decided to first examine the pyruvate decarboxylase-containing pathway by measuring the response of extracellular pyruvate and ethanol and the activities of pyruvate decarboxylase and alcohol dehydrogenase to the presence of thiamine in the media. Measurable quantities of ethanol are produced by H. maydis under both aerobic and anaerobic conditions (Table I). More extracellular ethanol is present under anaerobic than under aerobic conditions regardless of the nitrogen source used. The presence of thiamine in the media results in increased ethanol levels under both aerobic and anaerobic conditions; this is observed for all nitrogen sources except DL-alanine. A time course for the aerobic production of extracellular ethanol and pyruvate over a 5-day incubation period (Fig. 2) indicates that the level of ethanol is always greater when thiamine is present than when it is absent, although after the 2nd day of incubation both 0.1 and 1 .0 gg/ml thiamine are equally effective in stimulating ethanol production. In contrast to ethanol, the extracellular pyruvate levels are quite low. Over the entire incubation period, the pyruvate level decreases with increasing thiamine concentration. After 3 days of incubation, the ethanol and pyruvate concentations of the media were measured, and the tissue was transferred to fresh media of similar composition. After 6 hr of incubation in the fresh media, the ethanol and pyruvate concentrations were again determined. Both ethanol (Fig. 3A) and pyruvate (Fig. 3B) accumulate rapidly in the media, and the concentration response of these metabolites to thiamine after 6 hr is the same as after 3 days. Intracellular pyruvate levels of 3-

day-old tissue (Fig. 3C) respond to thiamine in a manner similar to the extracellular levels; attempts to detect intracellular ethanol were unsuccessful. Assays of alcohol dehydrogenase and pyruvate decarboxylase activities from 3-day-old tissue (Table II) reveal that pyruvate decarboxylase activity is high, and the activity increases with increasing thiamine concentrations of the media. Alcohol dehydrogenase activities are relatively low, and the enzyme is unaffected by the thiamine concentration of the culture media. An in vitro experiment demonstrated that thiamine-pyrophosphate and not thiamine-HCl is the form of thiamine which is effective in

240

stimulating pyruvate decarboxylase activity (Table II).

Nei-

ther form of thiamine has an effect on alcohol dehydrogenase activity. These data suggest that the quantity of pyruvate decarboxylase in the tissue increases as the thiamine-HCl concentration of the media increases and that this is not due merely to the activation of pre-existing enzyme. Thus, thiamine appears to act as an inducer or derepressor for the synthesis of its own apoenzyme; thiamine has no such effect on alcohol dehydrogenase. The effect of thiamine on pyruvate and ethanol levels can be explained by the effect of thiamine on pyruvate decarboxylase

220200

180-

10140-

10

activity.

so40

20

----ro4 0

1

2

3

4

5

Days of incubation FIG. 1. Growth of H. maydis after various periods of incubation in basal medium containing 0 (-*-), 0.1 (-x-), or 1.0 (- --- -) ,ugl ml thiamine-HCl. Each point represents the mean of six determinations, and the vertical bars represent the standard error.

When H. maydis is incubated in a thiamine-free medium, pyruvate decarboxylase activity is low and pyruvate accumulates intracellularly. Presumably, a proportion of this intracellular pyruvate pool "leaks" into the culture medium resulting in a drop in pH. When thiamine is present in the medium, pyruvate decarboxylase activity is increased resulting in an increase in the rate of conversion of pyruvate to acetaldehyde and a rise in the pH of the culture medium. Acetaldehyde is then reduced to ethanol in the presence of alcohol dehydrogenase, an enzyme which is not affected by thiamine; and ethanol accumulates in the medium.

Plant

~~

Physiol. Vol. 57, 1976

815

THIAMINE EFFECTS IN HELMINTHOSPORIUM

Table I. Ethanol Production by H. maydis under Aerobic and Anaerobic Conditions in Relation to Nitrogen Source and Thiamine Concentration of Media Cultures were previously incubated for 3 days in basal medium, pH 6.5, containing 0 or 1.0 ALg/ml thiamine-HCI. The tissue was theh harvested, washed, divided in half, and each half placed in fresh media. One of the two culture halves was flushed with nitrogen gas and then both halves were incubated an additional 6 hr. The media were then analyzed for ethanol using gas chromatography. Thiamine Concentration ()Ig/ml)

1.0

0

Nitrogen Source

Anaerobic

Aerobic

Anaerobic

Aerobic

)lmoles/g Dry Wt of Tissue + SE

L-Asparagine

DL-Alanine

54.2 + 12.4

347.9 4- 103.9

195.5 + 87.2

869.1 + 330.0

381.0 + 180.7

86.9

+

20.1

652.0

+

261.1

544.1

+

303.2

435.0 + 213.0

108.7 + 65.6

217.4 + 87.2

130.4 + 74.4

Ammonium Chloride

91.3 + 54.5

651.8 + 387.8

128.3 + 51.2

912.3 + 444.1

Ammonium Sulfate

87.2 + 45.5

325.0 + 177.7

261.0 + 121.5

566.o

Ammonium Citrate

106.3 + 66.9

540.2 + 243.0

320.8 + 130.0

870.2 + 479.1

L-Glutamic Acid

__

~

1

aI Nl--

Days

of

S.

I

_

-

2

221.8

I

~~~ _-

----

+

- 3

4 4

0

-

1

1.0

Thiamine (ug/mi) 5

incubation

FIG. 2. Ethanol ( ) and pyruvate (-- -) content of culture media containing 0 (@), 0.1 (x), or 1.0 (-) ,ug/ml thiamine-HCI after various periods of incubation with H. maydis. Each point represents the mean of six determinations, and the vertical lines represent the standard error.

The increase in the extracellular ethanol content under anaerobic conditions is probably due to an increase in the amount of NADH available for participation in the alcohol dehydrogenasecatalyzed reduction of acetaldehyde. When thiamine is also present, ethanol production is increased both because of the NADH effect on alcohol dehydrogenase and the thiamine effect on pyruvate decarboxylase. Thiamine has been shown to increase ethanol (1, 3, 4, 6, 14, 15) and decrease pyruvate (2, 20) levels in other fungi. In Saccharomyces cerevisiae it has also been demonstrated that pyruvate decarboxylase activity increases when thiamine is added to the medium (16). In this latter case, there is evidence that thiamine induces the synthesis of pyruvate decarboxylase (21, 22). Other examples of coenzyme induction have been demonstrated in other organisms (8, 9). The studies presented here lend support to the evidence for coenzyme induction by thiamine. There is no equimolar relationship between the decrease in

0

0.1

1.0

Thiamine (ug/mi)

FIG. 3. Effect of thiamine-HCl on extracellular ethanol (A) and extracellular pyruvate (B) produced by H. maydis after 3 days (0) and 6 hr (E) incubation and on the level of intracellular pyruvate (C). After 3 days incubation, tissue was assayed for intracellular pyruvate. The media from other 3-day-old cultures were assayed for extracellular ethanol and extracellular pyruvate; and the tissue from these cultures was washed, transferred to fresh media, and incubated an additional 6 hr. The ethanol and pyruvate contents of these media after this relatively short incubation period were then determined. Each bar represents the mean of six replications, and the vertical lines represent the standard error. pyruvate and the increase in ethanol concentrations upon the

addition of thiamine. Also, the ethanol levels are approximately five times the pyruvate levels. These observations suggest that pyruvate is metabolized more rapidly than ethanol in H. maydis, and thus pyruvate does not accumulate in either the tissue or the media to any great extent. This involvement of thiamine in an alcoholic fermentation pathway in H. maydis provides information which may be useful in studying thiamine inhibition of sporulation. For example, the thiamine-stimulated accumulation of ethanol in the medium represents a loss of carbon from pathways which could be critical for sporulation. Also, the relatively low activity of alcohol dehydrogenase compared with pyruvate decarboxylase may be indicative of the thiamine-mediated buildup of acetaldehyde which could be inhibitory to both growth and sporulation; Garraway (unpub-

816

EVANS AND GARRAWAY

Plant Physiol. Vol. 57, 1976

Table II. Activities of Pyruvate Decarboxylase and Alcohol Dehydrogenase from H. maydis Tissue in Relation to both Thiamine-HCI Content of Culture Media and Form of Thiamine in Enzyme Assay Reaction Mixture The tissue was incubated for 3 days in basal medium containing different concentrations of thiamine-HCl. Enzyme preparations of both pyruvate decarboxylase (PD) and alcohol dehydrogenase (ADH) were made, and the activities of each were determined in the presence of no thiamine derivative, 7.5 mm thiamine-pyrophosphate (thiamine-PP), or 7.5 mm thiamine-HCl. Other than this modification of the type of thiamine derivative present, the procedures for these assays are those described under "Methods and Materials." Thiamine-HC1 Content of Medium (jg/mi) Enzyme

Thiamine Derivative

0.1

0

1.0

nmoles NADH oxidized/min/mg protein + SE PD

None

31 +

PD PD ADH

4.

5.

6. 7. 8. 9.

5

50

+ 11

53

18

Thiamine-HC1

28 +

5

25 +

6

36 +

9

None

4+

2

8+

3

7+

2

ADH

Thiamine-PP

6+

1

6+

1

5+

0

ADH

Thiamine-HC1

8+

3

5+

0

6+

0

LITERATURE CITED

2. 3.

27 +

Thiamine-PP

lished data) has shown that exogenously applied acetaldehyde, but not ethanol, inhibits sporulation in H. maydis. On the other hand, the stimulation of pyruvate decarboxylase by thiamine suggests that other thiamine-pyrophosphate-dependent enzymes may likewise increase in activity resulting in the stimulation of certain pathways which could be inhibitory to sporulation. The results presented here provide information which may be useful in a further examination of the biochemical and physiological roles of thiamine in H. maydis.

1.

2

+

CHIAO, J. S. AND W. H. PETERSON. 1956. Some factors affecting the inhibitory action of thiamine on the growth of Saccharomyces carlsbergensis. Arch. Biochem. Biophys. 64: 115-128. COCHRANE, V. M. 1958. Physiology of Fungi. John Wiley & Sons, New York. DAMMAN, E., 0. T. RonNt, AND F. F. NORD. 1938. Enzymatische Umsetzungen durch Fusarium graminearum Schwabe (Gibberella saubinetti), zugleich Beitrag zur Wirkungsweise der Blausure und des Vitamins B,. Biochem. Z. 297: 185-202. EspoSITO, R. G., H. GREENWOOD, AND A. M. FLETCHER. 1962. Growth factor requirements of six fungi associated with fruit decay. J. Bacteriol. 83: 250-255. EVANS, R. C. AND M. 0. GARRAWAY. 1974. Effect of thiamine on sporulation and growth of Helminthosporium maydis race T and on pyruvate and ethanol production. Plant Physiol. 53: S-48. FOSTER, J. W. 1949. Chemical Activities of Fungi. Academic Press, New York. GARRAWAY, M. 0. 1973. Sporulation in Helminthosporium maydis: inhibition by thiamine. Phytopathology 63: 900-902. GREENGARD, 0. AND M. GORDON. 1963. The cofactor-mediated regulation of apoenzyme levels in animal tissue. J. Biol. Chem. 238: 3708-3710. HAMMEL, C. L. AND S. P. BESSMAN. 1966. Heme stimulation of globin synthesis in a cell-

83+ 10

125 + 22

free system. Science 152: 1080-1082. 10. KORNBERG, A. 1955. Lactic dehydrogenase in muscle. Methods Enzymol. 1: 441-443. 1 1. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 12. MAGASANIK, B. AND H. E. UMBARGER. 1950. The separation and identification of ketoacids by filter paper chromatography. J. Am. Chem. Soc. 72: 2308-2309. 13. NEISH, W. P. 1957. a-Keto acid determinations. In: D. Glick, ed., Methods of Biochemical Analysis, Vol. V. Interscience Publishers, New York. pp. 107-179. 14. OURNAC, A. 1969. Influence de l'origine de la thiamine (levure ou milieu) sur la vitesse de la fermentation alcoolique. Ann. Technol. Agric. 18: 187-198. 15. SCHOPFER, W. H. AND M. GUILLOUD. 1945. Recherches experimentales sur les facteurs de croissance et le pouvoir de syntheses de Rhizopus cohnii Berl. et de Toni (Rhizopus suinus Neilson). Z. Vitaminforsch. 16: 181-296. 16. SUOMOLAINEN, H. AND E. OuRA. 1959. Changes in the decarboxylase activity of baker's yeast during the growth phase. Biochim. Biophys. Acta 31: 115-124. 17. THEN, R. AND F. RADLER. 1970. Regulation der Acetaldehydkonzentration in Medium wahrend der alkoholoschen Garung durch Saccharomyces cerevisiae. Arch. Mikrobiol. 72: 60-67. 18. ULLRICH, J., J. H. WlIrORF, AND C. J. GUBLER. 1966. Molecular weight and coenzyme content of pyruvate decarboxylase from brewer's yeast. Biochim. Biophys. Acta 1 13: 595604. 19. VALLEE, B. L. AND F. L. HOCH. 1955. Zinc, a component of yeast alcohol dehydrogenase. Proc. Nat. Acad. Sci. U. S. A. 41: 327-338. 20. WIRTH, J. C. AND F. F. NoRD. 1942. Essential steps in the enzymatic breakdown of hexoses and pentoses. Interaction between dehydrogenation and fermentation. Arch. Biochem. 1: 143-163. 21. WITT, I. AND L. HEILMEYER. 1966. Regulation of pyruvate decarboxylase (EC 4.1.1.1) by coenzyme induction in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 25: 340-345. 22. Wln, 1. AND B. NEUFANG. 1970. Studies on the influence of thiamine on the synthesis of thiamine pyrophosphate-dependent enzymes in Saccharomyces cerevisiae. Biochim. Biophys. Acta 215: 323-332.