Developmental regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa

DEVELOPMENTAL BIOLOGY 50, 122-133 (1976)

Developmental Regulation of Nicotinamide Adenine Dinucleotide (Phosphate) Glycohydrolase in Neurospora crassa

ROBERT E. NELSON,’ CLAUDE P. SELITRENNIKOFF,~ AND RICHARD W. SIEGEL

Department of Biology, University of California’, Los Angeles, California 90024

Accepted December 8, 1975

The formation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase [NAD(P)- ase; EC 3.2.2.61 in Neurospora crassa was found to be both spatially and temporally programmed. Ascospores were devoid of the enzyme. Vegetative hyphae contained little or no NADase activity. During the differentiation of aerial cell types (aerial hyphae and macroconidia), the specific activity of the enzyme increased by at least three orders of magnitude. Although transiently associated with young aerial hyphae, the enzyme became an integral and stable part of the mature macroconidia. NAD(P)ase could also be “derepressed” under conditions that permitted aerialogenesis in the absence of conidiation. The increase in the specific activity of NAD(P)ase during cell differentiation required concomitant RNA and protein synthesis; in vitro mixing experiments revealed no cell-specific activators or inhibitors of enzyme activity. The temperature-critical period for the in vitro inactivation of a temperature- sensitive enzyme variant was restricted to the period of actual enzyme expression.

The data reported in this paper combined with data reported in a previous paper (Nelson et al., 1975b) underscore an important distinction in studies of development, namely, developmental regulation of a macromolecule versus regulation of development by a macromolecule. This paper provides evidence that NAD(P)ase is developmentally regulated. The previous paper provides evidence that the appearance of this enzyme need not regulate development.

INTRODUCTION

Differential gene activity is widely accepted as a causa sine qua non for cell differentiation. In several cases it has been concluded that a change in enzyme activity during cell differentiation requires differential gene activity (Brown and David, 1969; Tomkins and Martin, 1970; Wright, 1973). However, it is imperative to distin- guish between two questions regarding such correlations between enzyme activity and cell differentiation: (1) Is the change in enzyme activity mandatorily connected to cell differentiation?, i.e., is the enzyme developmentally regulated? and (2) Is cell differentiation mandatorily connected to the change in enzyme activity?, i.e., is the change in enzyme activity critical for cell differentiation? We have recently pointed

’ Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wis- consin 53706.

L Present address: Department of Zoology, Uni- versity of Wisconsin, Madison, Wisconsin 53706.

out the suitability of Neurospora crasssa for such inquiries (Nelson et al., 1975a). The asexual life cycle of N. crussa consists of but three distinct cell types, vegetative hyphae, aerial hyphae, and macroconidia, whose sequential appearance can be brought under strict experimental control.

In a previous paper (Nelson et al., 1975b), we explored Question 2 above with respect to nicotinamide adenine dinucleotide (phosphate) glycohydrolase [NAD(P)- ase; EC 3.2.2.61. In this paper we explore Question 1 with the same enzyme. Pre- vious information suggested that NAD(P)ase is developmentally regulated in N. crassa; enzyme activity has been shown to be associated with the morpho- genesis of aerial cell types, i.e., aerial hyphae and macroconidia (Zalokar and Coch- rane, 1956; Urey, 1971). We pursue this suggestion further by taking advantage of the ability to block the development of particular cell types (with mutation or en- vironmental manipulation) and by utiliz-

122 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

NELSON, SELITRENNIKOFF AND SIEGEL

ing a strain which produces heat-labile NAD(P)ase.

MATERIALS AND METHODS

Chemicals. Actinomycin-D (Dactinomy- tin) was purchased from General Biochem- icals. Cycloheximide was purchased from Nutritional Biochemicals. Nicotinamide adenine dinucleotide (@NAD) and Tween 80 (polyoxyethylene sorbitan mono-oleate) were purchased from Sigma Chemical Company. [2-‘4C]Uridine was purchased from SchwarziMann.

Strains and culture conditions. Strain 74-OR23-1 (FGSC 987; Oak Ridge Na- tional Laboratory wild-type) and strain FGSC1648 [mo (morphological; B8)l were obtained from the Fungal Genetics Stock Center. Strains UCLA 503 Lacon-3 (ace- nidial) 1, DLP91 (acon-2)) UCLA37 [csp-l (conidial separation)], and FS591 (csp-2) contain single-gene mutations that block the process of macroconidiation; the ori- gins of these strains have been described previously (Matsuyama et al., 1974; Seli- trennikoff et al., 1974). Standard methods were used for the maintenance and storage of these strains (Davis and de Serres, 1970). In order to isolate large quantities of dry, unwashed conidia, cultures were also prepared in 60 x 20-mm petri dishes containing agar-solidified Vogel medium N (Vogel, H. J., Microb. Genet. Bull. 13: 42- 43,1956) plus 1.5% (w/v) sucrose and main- tained at 20°C in continuous light. After 2 weeks, cultures were opened, inverted over glassine paper, and tapped to collect mature conidia.

In standard cultures, the growth of vegetative hyphae and the differentiation of aerial hyphae and conidia are for the most part coincident. In order to study these processes separately, cultures were prepared in which vegetative growth and asexual differentiation were completely separated in time (cf., Siegel et al., 1967; Nelson et al., 197513). Vegetative hyphae were cultured from conidia (1 x 10’ per flask) in 250-m] Erlenmeyer flasks con-

Developmental Regulation in Neurospora 123

taining 50 ml of Vogel medium N plus 1.5% (w/v) glucose and 0.01% (v/v) Tween 80. Cultures were incubated without agitation at 25°C for 90 hr or at 37°C for 66 hr. Under these conditions, mycelial pads of vegetative hyphae formed at the liquid-air interface of the medium without the concomitant formation aerial cell types. To permit aerial-cell differentiation, pads were washed free of growth medium, soaked in 0.05 M potassium phosphate buffer (pH 6. l), and cut into mycelial rings (8-cm o.d., 6.8-cm i.d.). Beforehand, 150 x 25-mm petri dish bottoms were filled with 75 ml of buffer and placed in 20 x 8-cm finger bowls. Stainless-steel screens (15 x

10 cm) were covered with g-cm wide strips of buffer-soaked filter paper and placed across the open dishes. The ends of the strips were folded over the l&cm edges of the screens and extended down into buffer. Mycelial rings were placed in separate finger bowls on the centers of the elevated filter paper strips. The finger bowls were covered with glass plates affixed with rub- ber sealing rings and incubated at 25 and 37°C under 300 fc of artificial daylight. In some experiments, the apparatus was modified in one of two ways: (1) The glass cover that normally seals the finger bowl was elevated 3 mm above the rim of the bowl. This modification resulted in the absence of development of all aerial cell types, but did not abolish the competence of the vegetative hyphae to form these cells under appropriate conditions (vegetative hyphae starved for 8 hr in “open” bowls could produce normal quantities of aerial hyphae and conidia when starved in sealed bowls for an additional 12 hr). (2) Covered finger bowls were flushed with nitrogen gas and sealed. By maintaining the oxygen content of the sealed bowls below 2%, only aerial hyphae were formed by the starving mycelial ring; no conidia could be detected after 12-hr incubation.

Analytical techniques. Glucose utiliza- tion was monitored with the Glucostat reagent (Worthington Biochemical Corp.,

124 DEVELOPMENTAL BIOLOGY VOLUME 50, 1976

1972). The protocol for acid inactivation of extracellular enzymes in situ (Fig. 1, legend) was modified from that of Scott and Metzenberg (1970). Protein was determined with the Folin-Ciocalteu phenol reagent (Lowry et al., 1951) or biuret reagent (Schneider, G. et al. (1969), Neurosp. Newsl. 14, 10-11).

Dry, unwashed conidia were collected from cultures in finger bowls by “dusting” the conidia off the aerial hyphae and onto glassine paper strips with a forced air stream. To calculate the dry weight of the conidia, samples were suspended in water and the concentration of conidia determined with a haemocytometer. Aliquots of the suspensions were then dried to constant weight on preweighed Millipore fil- ters. The dry weights of conidia from cultures of the same age and from cultures of different ages were constant (3.1 ? 0.1 mg per 1 x lo8 conidia).

For the determination of the dry weight of each cell type, cultures (in finger bowls) were fractionated as follows. Aerial cell networks were severed from underlying vegetative mycelia with the edge of a spat- ula and collected on Paratilm. Aerial cell material and “stripped” vegetative mycelia were dispersed in water and the dry weight and number of conidia in each suspension were determined as described above. The dry weight of aerial hyphae was taken to be the dry weight of the total aerial cell mass minus the dry weight of conidia recovered in the aerial cell mass. Because the fractionation procedure causes displacement of some conidia from the aerial cell network to the underlying vegetative hyphae, the dry weight of the vegetative cell mass was corrected for the weight of any displaced conidia.

Methods for the preparation of cell washes, breis, and extracts were as described in Nelson et al. (1975b). The NADase activity NAD(P)ase was determined by the method of Colowick et al. (1951). Enzyme activities are reported in micromoles of NAD hydrolyzed per minute

at 25°C. Specific activities are calculated in units per milligram (dry weight) of cells. The term “patent” refers to enzyme activity that can be detected in whole cells; “cryptic” refers to any additional enzyme activity that can be detected after disruption of cells. Because the NADase activity of conidia from cultures in linger bowls was found to be constant and independent of culture age (see Results), the apparent NADase activities of aerial hyphae and vegetative hyphae were readily corrected for the enzyme activity of any normally or spuriously associated conidia.

RESULTS

Subcellular localization of NAD(P)ase. In order to accurately estimate changes in total enzyme activity which accompany development, the distribution of catalyti- cally active NAD(P)ase between the out- side and inside of cells was determined. Earlier studies suggested that N. crassa NAD(P)ase was probably an ectoenzyme (Zalokar and Cochrane, 1956; Stine, 1969).

Several lines of evidence indicate that most, if not all, of the conidial enzyme activity is situated external to cellular permeability barriers for NAD. Samples of unwashed conidia from strain 74-OR23-1 (wild-type) were found to be remarkably constant in specific NADase activity, 4.62 ? 0.34 unitsimg of dry conidia. A significant, but highly variable, proportion of total enzyme activity could be extracted from intact conidia by shaking conidia in phosphate buffer (22-92% of total enzyme activity with different lots of conidia). Yet, washed intact conidia and washed disrupted conidia differed very little in residual enzyme activity. Table 1 contains data from a typical experiment. Extraction of intact conidia with phosphate buffer removed 65% of the total enzyme activity. Washed, disrupted conidia exhibited 6.9% more activity than washed intact conidia, but this difference corresponded to only 2.4% of the total enzyme activity.

Another criterion that has been used to

NELSON, SELITRENNIKOFF AND SIEGEL Developmental Regulation in Neurospora 125

TABLE 1

LOCALIZATION OF NADASE ACTIVITY IN CONIDIA”

Sample Enzyme units from 10” co-

nidia

1. Supernatant from original suspen- 8.32 sion

2. Supernatant from the washed pel- 0.30 let of conidia

3. Washed conidia 4.34 4. Washed conidia disrupted in 4.66

Hughes press!’ 5. Supernatant from washed and dis- 2.00

rupted conidia 6. Total (lines 1 + 2 + 4) 13.28

‘I Dry harvested conidia from strain 74-OR-1 (wild-type) were suspended in cold 0.05 M phosphate buffer, pH 6.1, at a concentration of 1.5 x 10” cells/ ml and agitated for 1 hr at 0-4°C. The suspension was centrifuged at 10,000 g for 20 min. and the supernatant solution was assayed for enzyme activity. The conidial pellet was resuspended in additional cold buffer at 1.5 x lo!’ cells/ml, centrifuged as before, and the wash solution assayed. The washed conidia were again suspended in cold buffer at 1.5 x 10” cells/ml and a sample of these intact cells was assayed. An aliquant of this suspension was also passed through a Hughes press and assayed both before and after particulate material had been removed by centrifugation (10,000 g, 20 min).

” This treatment disrupted 86% of the cells.

help define the location of enzymes in Neu- rospora is the sensitivity of enzymes to inactivation in situ by dilute acid (Scott and Metzenberg, 1970). Intact conidia from strain 74-OR-1 were treated with 0.05 N HCl under conditions which did not reduce the viability of the conidia (97% survival). Both soluble and cell-bound NAD(P)ase were quickly inactivated (Fig. 1). Only 2.5% of the total enzyme activity remained refractory to the acid treatment. Clearly, if any cryptic compartment of NAD(P)ase exists in conidia, it must contain but a few percent of the active conidial enzyme.

NADase activity can also be detected in young conidiogenic aerial hyphae before the onset of conidiation (see below, and Urey, 1971). Vegetative cultures of strain 74-OR-1 were washed free of growth medium and permitted to form aerial hyphae for 4 hr. During this period, each 38-40 mg

of culture (dry weight) produced l-2 mg of aerial hyphae, 7-8.5 units of NADase activity, and no chains of proconidia or free conidia. Between 37-70% of the enzyme could be removed from intact hyphal cells with phosphate buffer. That fraction of the enzyme which was cell-bound yet accessi- ble to substrate in intact hyphae could not be directly measured as any attempt to disperse the hyphal network for enzyme assay resulted in significant cell lysis. Treatment of the hyphae with dilute acid resulted in protoplasmic fragmentation and loss of viability; hence, this method was also unsatisfactory for the determination of patent enzyme activity. After ex-

FIG. 1. Inactivation of NAD(P)ase in viable conidia by acid treatment. Dry-harvested conidia from strain 74-OR-1 (wild-type) were suspended in 0.05 N HCl at 0-4°C. After the indicated periods of acid treatment, samples were diluted in equal volumes of cold 0.048 N NaOH, and the pH of the suspensions was adjusted to 7.0 with additional dilute alakli. Zero-minute controls were prepared by suspending the conidia in 0.025 M NaCl at O-4%. After neutrali- zation the samples were centrifuged for 20 min at 1000 g, and the supernatants were assayed directly for soluble enzyme activity. The pellets were resuspended in 0.1 M phosphate buffer, pH 7.0, disrupted in a Hughes press, and asayed for residual enzyme activity. 0, soluble extracellular enzyme activity; 0, cell-bound enzyme activity.


haustive mechanical disruption of the hyphae, crude cell-wall fractions (washed 500 g pellets) still retained 16-20% of the total NADase activity. These results suggest that the “aerial” enzyme, like the conidial enzyme, is also located on the cell surface, external to cellular permeability bar- rier(s).

NADase level during the growth of vegetative hyphae. Vegetative hyphae of strain 74-OR-1 (wild-type) were cultured in unshaken liquid medium under conditions which did not permit the development of aerial cell types (Fig. 2, legend). Each 50 ml of culture had been inoculated with 2.5 x lo7 conidia, and thereby, 3.34 units of conidial NADase activity were introduced into each culture at the time of inoculation. Although 25% of this NADase activity immediately disappeared in the growth medium, the residual enzyme activity (units per culture) was relatively constant during 90 hr of vegetative cell growth (Fig. 2B). Moreover, when fresh cultures were rapidly agitated for 2 hr immediately following inoculation, 94-97% of the conidial NADase activity disappeared (Fig. 2B). No significant increase in NADase activity could be detected in these cultures during subsequent glucose catabolism even though the cellular mass in the cultures increased about 200-fold (Fig. 2A). In sharp contrast, when an identical sample of the conidial inoculum was incubated in distilled water (a condition which does not permit the outgrowth of conidia into vegetative hyphae), the associated NADase activity was stable for 90 hr (Fig. 2B). These observations indicated that the NAD(P)- ase detected in 90-hr-old vegetative cultures was simply that enzyme which had been introduced into the cultures at the time of inoculation.

NADase level during the differentiation of aerial cell types. The growth of vegetative hyphae in unshaken liquid culture resulted in the formation of a mycelial “pad” at the liquid-air interface of the growth medium. Pads of vegetative hy-

phae from 90-hr cultures of strain 74-OR-1 (wild type) were washed free of growth medium, soaked in phosphate buffer, cut into mycelial “rings,” and starved in a humid aerial environment within glass-covered finger bowls under artificial light. These conditions permitted the relatively synchronous development of sequential aerial cell types (Fig. 3A). Aerialogenesis was confined to the 5-hr period between the second and seventh hours following the onset of starvation. (Between the second and fourth hours, aerial hyphal growth could be monitored only by microscopic examination as the aerial hyphal mass was too small to be separated from the underlying vegetative mycelium); free macroconidia were detected in the aerial hyphae at the sixth hour and rapidly increased in number during the next 6-hr period. After incubation for 12 hr in the finger bowls, 30-35% of the initial vegetative cell mass had been converted into aerial cell types with less than a 10% loss in total cell dry weight.

In these “finger-bowl” cultures, the onset of NAD(P)ase accumulation was coincident with the onset of aerialogenesis. A fraction of the enzyme activity that was recovered with the vegetative hyphal mass of each culture could not be accounted for by the presence of displaced conidia (Fig. 3B). We believe that this fraction is confined to those portions of the aerial hyphae which could not be mechanically separated from the vegetative hyphae for the following reasons. (1) The aerial hyphae produced by cultures 2- to 3-hr old were too small to be removed from the mycelial rings and were unavoidably assayed as part of the vegetative hyphal cells. (2) In older cultures, the aerial cell networks were efficiently removed from above the surface of the vegetative mycelia, but the extensive “roots” of the aerial hyphae remained embedded within the vegetative hyphal masses. That this root system could contain all the NADase activity at- tributed to the vegetative hyphae is sup-

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‘4 10 20 30 40 50 60 70 80 90

Incubation (her)

FIG. 2. NADase level in unshaken cultures of vegetative hyphae. Cultures of strain 74-OR-1 (wild-type) were prepared in 250-ml Erlenmeyer flasks containing 50 ml of Vogel’s medium N plus 1.5% glucose and 0.01% Tween 80 and inoculated with 2.5 x 10’ conidia. Freshly inoculated cultures were either incubated directly at 25°C in dark moist chambers or shaken for 2 hr at 200 rpm and then left undisturbed in the dark chambers until harvested. In parallel, 2.5 x 10’ conidia were also incubated without agitation in 50 ml of distilled water. (A) 0, the dry weight of cultures in Vogel’s medium; m, glucose level in the culture medium. (B) & NADase level in conidia incubated in distilled water; 0, NADase level in cultures in Vogel’s medium; 0, NADase level in cultures in Vogel’s medium which were shaken during the first 2 hr of incubation.


2 4 b 10 12

Incubation (hrs)

FIG. 3. NADase level in three cell types during differentiation. Mycelial pads of vegetative hyphae of strain 74-OR-1 (wild-type) were prepared by growth in 250-ml Erlenmeyer flasks containing 50 ml of Vogel’s Medium N plus 1.5% glucose and 0.01% Tween 80. Cultures had been inoculated with 2.5 x 107 conidia and were incubated at 25°C in dark moist chambers without agitation. After 90 hr growth, pads were washed free of growth medium, soaked in phosphate buffer, cut into mycelial rings, and incubated in an aerial


ported by the following observation. When medium-free, buffer-soaked vegetative mycelial rings were starved in an aerial environment that would not permit the differentiation of aerial hyphae and conidia, NADase activity did not accumulate (Table 3, “open” culture). Thus, starving cultures which do not form aerial hyphae and are composed of only vegetative hyphae do not form detectable enzyme.

NADase activity was only transiently associated with conidiogenic aerial hyphae (Fig. 3B). The specific enzyme activity of this cell type was maximum (1.48 units/ mg dry weight) after cultures had devel- oped for 8 hr; by the twelfth hour, specific enzyme activity had dropped to 0.2 units/ mg dry weight of aerial hyphae. The maximum specific NADase activity of the aerial hyphae represented a ca. 700-fold increase over that of the vegetative hyphae at the onset of starvation.

Dry, unwashed conidia were collected from starving cultures at 2-hr intervals and were found to be similar in specific NADase activity, i.e., 4.54 +_ 0.63 units/mg dry weight; this activity represented a ca. threefold increase over the maximum specific NADase activity of the aerial hyphae and a 2000- to 2500-fold increase over the specific enzyme activity of the vegetative mycelium.

Accumulation of NADase activity during cell differentiation most likely reflects the attendant accumulation of enzyme molecules. Crude extracts of vegetative hyphae and conidiogenic aerial hyphae from strain 74-OR-l (wild-type) were mixed, incubated for various periods of time, and then assayed for NADase activity. As shown in Table 2, the enzyme activities of these preparations were additive. This indicated that the difference in the specific activities of these cell types was

TABLE 2

NADASE ACTIVITY IN MIXED CRUDE EXTRACTS

FROM VEGETATIVE HYPHAE AND CONIDIOCENIC

AERIAL HYPHAE OF STRAIN 74-OR-1 (WILD-

TYPE)“. *

Prein- cubation

(min)

Extracts’ NADase Activity

(units/ml)

Pre- Ob- dieted served

0

60

120

Conidiogenic aerial hyphae

Vegetative hyphae 1: 1 Mixture


Vegetative hyphae 1:l Mixture


Vegetative hyphae 1:l Mixture

- 12.95

- 0.01 6.48 6.55

- 13.19

- 0.01 6.60 5.97

- 11.50

- 0.04 5.77 5.45 -

” Vegetative hyphae were grown for 90 hr in 25°C in standard unshaken culture and harvested directly for enzyme extraction.

Ir Vegetative hyphae were grown for 90 hr at 25°C in standard unshaken culture. Medium-free, buffer- soaked hyphae were allowed to differentiate in the standard aerial environment for 7.5 hr, and the aerial hyphal network was harvested for enzyme extraction.

’ Freshly harvested samples were ground in acid- washed sand with a chilled mortar and pestle. The resulting homogenates were suspended in 0.1 M phosphate buffer, and large particulate material was removed by centrifugation at 2000 g for 20 min. Crude extracts (supernatant solutions) were assayed for enzyme activity separately and in 1:l mixture both before and after preincubation at 33°C. Crude extracts were not dialyzed in this experiment. The protein concentration of all extracts was 2.80 + 0.22 mgiml.

not due to cell-specific dissociable activators or inhibitors of the enzyme, and also suggested that the vegetative hyphae do not contain a NAD(P)ase zymogen which is activated by maturation enzymes that

environment which permitted the formation of aerial hyphae and conidia. (A) dry weight of each cell type: A, vegetative hyphae; 0, aerial hyphae; 0, conidia. (B) NADase level in each cell type: A, vegetative hyphae; 0, aerial hyphae; 0, conidia.


are normally confined to the aerial hyphae.

Cycloheximide (0.8-1.0 pglml) has been shown to inhibit protein synthesis by >90% within 60 min in medium-free cultures of N. crassa (Horowitz et al., 1970). When cycloheximide (1 pg/ml) was added to cultures of strain 74-OR-1 (in finger bowls) 1 hr before the onset of aerialogenesis, subsequent development of aerial hyphae and conidia and NAD(P)ase accumulation were completely inhibited; this ef- fect was reversible. When actinomycin-D (20 pg/ml) was added to cultures 1 hr before the onset of aerialogenesis, incorpora- tion of [14Cluridine (3.8 x 10e5 M; 53 mCi/ mmol) into trichloroacetic acid-insoluble material was inhibited by 66% within 90 min; subsequent aerial-cell differentiation and NAD(P)ase accumulation were se- verely depressed (i.e., only microscopic evidence of aerial hyphae and proconidia and a 20-fold reduction in the accumulation of NADase activity were apparent). These observations indicate that NAD(P)ase accumulation depends on net protein and RNA synthesis within the period of aerial cell formation.

Previously, we described the identifica- tion of nada(62ts), a structural gene mutation which specifies a temperature-sensitive NAD(P)ase; the activity of this enzyme was temperature-sensitive in. uiuo as well as in vitro (Nelson et al., 1975b). The temperature-critical period for the in viva

inactivation of the mutant enzyme was found to be coincident with the developmental period of enzyme expression, that is, within the period of aerial-cell differentiation. Increasing the incubation temperature during vegetative growth had no ef- fect on enzyme expression during subsequent cell differentiation at low temperature (cf., Table 7 in Nelson et al., 1975b).

Collectively, the foregoing observations support the conclusion that the developmental expression of NAD(P)ase depends on the de novo synthesis and accumulation of enzyme molecules during aerial-cell differentiation rather than the “unmasking” of preformed enzyme.

The relationship of NAD(P)ase to cell differentiation. With the isolation of structural gene mutants, we have been able to show that the catalytic activities of NAD(P)ase are not required for the formation of aerial hyphae and conidia or for conidiospore germination (Nelson et al., 1975b). On the other hand, accumulation of enzyme activity does appear to be developmentally regulated in wild-type cultures (Table 3). When vegetative hyphae of strain 74-OR-1 (wild-type) were starved for 9 hr in an aerial environment which permitted neither aerialogenesis nor conidi- ogenesis (“open” cultures), NADase level increased only slightly. But under conditions which permitted the development of aerial hyphae while prohibiting conidio- genesis, the NADase level rose 500-fold, an

TABLE 3

NADASE LEVEL IN MEDIUM-FREE CULTURES WHICH WERE PERMITTED TO FORM AERIAL HYPHAE AND CONIDIA, AERIAL HYPHAE ONLY, OR No AERIAL CELL TYPES”

Type of culture Vegetative cell mass (mg)

Aerial Cell mass (mg)

Number of conidia NADase level per culture (units per cul-

ture)

Standard 25.6 9.2 7 x 10’ 26.67 Flushed with N, 26.4 7.9 <lo” 17.62 “Onen” 35.6 0 <lo@ 0.12

a Vegetative hyphae of strain 74-OR-1 (wild-type) were grown for 90 hr at 25°C in standard unshaken culture. Medium-free, buffer-soaked mycelial rings were starved at 25°C in standard or modified finger bowls (see Materials and Methods). After 9 hr, the cultures were harvested for the determinations of dry cell masses, numbers of conidia, and enzyme activity. The NADase level of the cultures at the onset of starvation was 0.035.

6 Limit of detection.


TABLE 4

NADASE ACTIVITY IN CULTURES OF DEVELOPMENTAL MUTANT@

Strain Stage at which develop- Vegeta- Aerial Number of NADase ment is blockedb tive cell cell mass conidia per level (units

mass (mg) culture per cul- (mg) ture)

74-OR-1 (wild-type) Completes development 23 15 2 x 108 38.2 UCLA 37 ksp-1) Chains of proconidia; leaky 22 17 1 x 10” 34.5 FS 591 &p-2) Chains of proconidia; leaky 21 13 4 x lo” 50.4 UCLA 503 (acon-3) Aerial hyphal tips contain- 27 8 < 103” 30.1

ing nuclei DLP 91 (acon- Aerial hyphal tips without 28 3 Cl03 12.9

nuclei FGSC 1648 (molB81) - 34 - 1’ <lOY’ 1.9

u Vegetative hyphae were grown for 90 hr at 25°C in standard unshaken culture. Medium-free, buffer- soaked mycelial rings were starved in a humid aerial environment within sealed finger bowls at 25°C under artificial daylight. After 12 hr, cultures were harvested for the determinations of dry cell masses, numbers of conidia, and enzyme activity.

b For a discussion of the temporal sequence of stages in the process of aerial-cell differentiation, see Matsuyama et al., 1974.

( Limit of detection. ” Cultures of strain FGSC 1648 produce only microscopic amounts of aerial hyphae that cannot be

separated from the vegetative hyphae.

increase comparable with that of similarly aged cultures that formed both aerial hyphae and conidia. These data strongly suggest that aerialogenesis is a sufficient condition for the accumulation of NAD(P)ase. A further examination of this point was sought by genetic, rather than environ- mental, manipulations and is described below.

A number of aconidial mutants whose vegetative properties are not radically al- tered from those of wild-type have been collected in our laboratory. Characteriza- tion of several single-gene mutants also supports the idea that NAD(P)ase is developmentally regulated (Table 4). (1) The two mutants that are blocked after the formation of proconidia yield wild-type levels of NADase activity. (2) In mutants that are blocked prior to the formation of proconidial chains, the NADase level is directly proportional to the extent of aerial hyphal elaboration.

DISCUSSION

The accumulation of NAD(P)ase in N. CMSSU is developmentally regulated. No net increase in enzyme activity can be de-

tected during the growth of wild-type vegetative hyphae. During “programmed starvation” (Wright, 19731, vegetative hyphal growth is replaced by the elaboration of conidiogenic aerial hyphae, and is char- acterized by the net accumulation of active enzyme molecules within these newly formed cells. In wild-type cultures which proceed to fruition, NAD(P)ase is only transiently associated with aerial hyphae, but is a long-lived component of dry mature conidia.

Four points of interest with respect to the developmental regulation of NAD(P)- ase have come out of the above analyses. (1) The enzyme appears to be sharply lo- calized in the fully differentiated asexual phase of N. crassa. The vast majority, and possibly all, of the active enzyme is found in the macroconidia even though this cell type accounts for less than 25% of the dry mass of the organism (Fig. 3). By contrast, no enzyme activity can be detected in the corresponding cell type (the ascospore) of the sexual phase of N. crassa (data not shown). (2) Accumulation of enzyme activity during differentiation appears to be due to de nouo synthesis. Inhibitor studies

al., 1975b). No difference in the life cycles of wild-type and NAD(P)ase-less mutants has been detected. In particular, the rate and extent of the differentiation of aerial hyphae and conidia, which appear during the period when NAD(P)ase accumulates in wild-type, are indistinguishable in these strains. Thus, NAD(P)ase is a developmentally regulated enzyme whose bio- logical significance remains an enigma.


suggest that net RNA synthesis and net protein synthesis are required for the accumulation of enzyme activity. Analysis of mixed cell extracts does not indicate the presence of cell-specific activators or inhibitors of the enzyme (Table 2). Tempera- ture-shift experiments utilizing a mutant that produces a heat-labile NAD(P)ase have established that the enzyme is not synthesized during vegetative growth in a masked form which is subsequently converted to an active form during aerial-cell differentiation (Nelson et al., 1975b). (3) Starvation per se is not a sufficient cue for NAD(P)ase accumulation. In a non-nutri- tive liquid environment, vegetative hyphae do not produce NAD(P)ase (Urey, 1971). More significantly, vegetative hyphae that are starved in an aerial milieu also do not yield increased enzyme activity (Table 3). (4) Aerialogenesis appears to be a sufficient condition for the accumulation of NAD(P)ase. Data in both Tables 3 and 4 underscore the fact that enzyme activity does increase under conditions (environ- mental or genetic) which permit only aerialogenesis.

We wish to thank Dr. David Sonneborn for many helpful suggestions for the preparation of this man- uscript. We also wish to thank Ms. Teryl Chandler for her technical assistance.

These investigations were supported in part by a UCLA Medical Sciences Research Fund to P. T. Cohen, and NSF grants to R. W. S.

REFERENCES

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There is one report in the literature which, at first glance, disagrees with observations reported in this paper. Stine (1969) observed a steady increase in the apparent NADase activity of vegetative hyphae after shaking liquid cultures had been incubated for 18 hr at 25°C. We also observed a precipitous rise in the enzyme level of such cultures. Yet, microscopic examination of the cultures revealed the presence of short chains of proconidia. We suggest, therefore, that the rise in the NADase activity in aged shake-cultures is due to the production of NAD(P)ase by the aerial-cell contingent.

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Developmental regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa