We were unable to find transient changes in the amount of cAMP or cGMP that had been proposed to mediate the light-growth response in sporangiophores of Phycomyces blakesleeanus

Brandt, Michaela

Leutwiler, Leslie S.

English

PubMed

Caltech Authors - Main

Vol. 153, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 555-557
0021-9193/83/010555-03$02.000/
Absence of Significant Light-Induced Changes in cAMP
Levels in Sporangiophores of Phycomyces blakesleeanus
LESLIE S. LEUTWILER* AND MICHAELA BRANDT
Division ofBiology 156-29, California Institute of Technology, Pasadena, California 91125
Received 14 June 1982/Accepted 16 September 1982
We were unable to find transient changes in the amount of cAMP or cGMP that
had been proposed to mediate the light-growth response in sporangiophores of
Phycomyces blakesleeanus.
Sporangiophores (SPHs) of Phycomyces bla-
kesleeanus show several responses to blue light,
among which are the light-growth response (a
transient increase in growth rate) and phototro-
pism. Both responses are sensitive over a wide
range of light intensities (10-9 to 6 W/m2) and
show action spectra which implicate a flavin
photoreceptor (4, 5, 10). It has been reported (2,
3) that within 0.5 min after the onset of illumina-
tion, the cAMP content of the SPH decreases by
more than 60% and then returns within the next
2 min to its original value. Cohen (2) proposed
that this modulation is an important link in the
transduction chain between absorption of light
and the transient change in growth rate which
occurs 4 to 5 min later.
Since the light-growth response of the SPH is
well characterized (7-9) and behavioral mutants
are available (1), we initiated this study to inves-
tigate the biochemical events leading to the light
responses. We began by looking for changes in
levels of cAMP or cGMP that could be correlat-
ed with a step up in light intensity. We were
unable to find such changes, even under condi-
tions similar to those reported by Cohen (2, 3).
We did find that it was extremely important to
standardize both growth and experimental con-
ditions. One modification in the method of Co-
hen was the synchronization of SPH growth.
Otherwise, we could not define any specific
differences in methodology that would account
for the different results obtained.
The standard wild type of P. blakesleeanus
used in this work was strain NRRL 1555(-)
from the Northern Regional Research Labora-
tory, U.S. Department of Agriculture, Peoria,
Ill. To obtain a synchronized population of
SPHs, the method of Russo (11) was used.
Approximately 400 heat-shocked (5 to 10 min at
47°C) spores were spread on each of several
petri plates (100 by 15 mm) containing 25 ml of
potato dextrose agar and thiamine (0.5%). The
plates were placed under 1-liter beakers in a
shallow pan of water so that no gas exchange
would take place. After 60 h of incubation at
19°C in the dark, the mycelia were illuminated
with white light (two 24-W Cool White fluores-
cent lamps; total intensity, 1 W/m2). SPHs de-
veloped to stage I within the next 36 h. The
beakers were then removed, and the SPHs de-
veloped to stage IVb within approximately 24 h,
at which time they measured between 3.5 and
4.0 cm in length.
Twelve plates each containing about 1,000
synchronized stage IVb SPHs were placed in an
illumination chamber and dark adapted for 40
min. Three plates were harvested in the dark,
and the light was switched on (t = 0). Three
plates were harvested at 0.5, 0.75, and 5 min.
The blue light source consisted of 16 40-W
tungsten-filament incandescent bulbs as de-
scribed by Jayaram et al. (6).
The SPHs from three plates (approximately 1
g [total fresh weight]) were harvested by plung-
ing them into liquid nitrogen. The upper 2.4 cm
was cut off, ground to a fine powder in a mortar
containing liquid nitrogen, and homogenized in
10 ml of ice-cold 0.6 N perchloric acid to which
trace amounts of [3H]cAMP (11,100 dpm; 0.3
pmol) and [3H]cGMP (11,100 dpm; 0.6 pmol)
had been added (amounts sufficiently small to be
undetectable in the assay). The mixture was
centrifuged, and the pellet was dried in a 70°C
oven for approximately 24 h and then weighed.
The supernatant fraction was neutralized to pH
6.0 to 6.2 with 6 M KOH, incubated on ice for 1
h, and centrifuged to remove the KCl04 precipi-
tate. Part of the supernatant fraction (1 ml of
crude extract) was frozen for future assay.
The rest of the supernatant fraction was load-
ed on a Bio-Rad AG 1-X8 column (200 to 400
mesh, formate form, 10 cm). The loaded column
was washed with 15 ml of distilled water fol-
lowed by 30 ml of 0.1 N formic acid (pH 2.3).
The cAMP fraction was eluted with 15 ml of 2 N
formic acid (pH 1.5). The cGMP fraction was
eluted with 35 ml of 4 N formic acid (pH 1.1).
Both fractions were lyophilized and redissolved
555
TABLE 1. cAMP Extracted from four batches of SPHs with or without 500 pmol of authentic cAMP and
from the extraction solution containing 500 pmol of authentic cAMP
cAMP recovery (pmol) for following batch: % Recovery
Extract SPHs SPHs + cAMP cAMP
extraction Extraction
1 2 3 4 solution Batches 1-4 solution
Crude 364 359 692 556 481 52 96
Pure 431 402 939 812 524 92 105
in 1 ml of 0.05 M sodium acetate buffer, pH 6.2.
Recovery for cAMP was 80% and for cGMP was
65% as judged from the 3H content of the
fractions.
Duplicate assays for each cyclic nucleotide
were performed on both the crude and purified
extracts, using the radioimmunoassay method of
Steiner et al. (12) with kits supplied by New
England Nuclear Corp.
A standard curve was prepared relating true
dry weight to the weight of the dried perchloric
acid pellet. This modification was used because
of the necessity for immediate acid fixation of
the samples after harvest. Therefore, cAMP
levels are reported as picomoles per gram (dry
weight) of the perchloric acid precipitate. This
represents approximately 85% of the true dry
weight and about 10% of the fresh weight.
To check for cAMP authenticity, purified
samples were treated with phosphodiesterase
and chromatographed with authentic samples of
cAMP and cGMP. To check the accuracy of the
procedure, unlabeled internal standards of
cAMP were added to SPHs before extraction.
Four batches of SPHs (three plates of SPHs per
batch) were harvested: batches 1 and 2 were
treated in the normal way; batches 3 and 4 were
mixed and divided into two equal fractions. A
500-pmol amount of authentic cAMP was added
to each batch. A similar amount of cAMP was
added to a sample of the extraction solution. The
results for both the crude and purified extracts
are shown in Table 1.
Assuming that X = amount of cAMP in one
batch of SPHs before extraction and a = percent
recovery of cAMP, the amount ofcAMP present
in batch 1 and batch 2 after extraction is:
aX = A (1)
Similarly, the amount of cAMP in batch 3 and
batch 4 is:
a(X + 500) = B (2)
Substitution of equation 1 into equation 2 gives:
B-A
Ot5=
500 (3)
The mean value for the amount ofcAMP detect-
ed in the crude extracts from batches 1 and 2
was 362 pmol (A) and for batches 3 and 4 was
624 pmol (B). This implies (equation 3) that the
amount of cAMP recovered from the crude
extract was 52%. A similar calculation for the
purified extract implies a 92% recovery. Since
recovery when only authentic cAMP was pre-
sent was 96%, it appears that something in the
crude extract is inhibiting the binding of cAMP
to the antibody. Therefore, it was necessary to
purify the cAMP before assay.
This experiment also gave an indication of the
variation inherent in the procedure. cAMP val-
ues for identical samples (batches 3 and 4)
showed standard deviations of the mean of
slightly more than 10% for the crude and 7% for
the pure extracts. Because of this variation, it is
difficult to draw any conclusions from the results
of one experiment, unless differences in cAMP
content are large.
We found no significant change in cAMP
concentration after a step up of light intensity
from the dark to a level above that required to
saturate the light-growth response. Table 2
shows the results of nine separate experiments.
It is possible that the regulation of cyclic
nucleotide levels takes place only in the growing
zone of the SPH (located in the 3-mm area
directly below the sporangium). Evidence for
this proposal comes from the cytochemical lo-
calization of adenylate cyclase only in the grow-
ing zone and its absence in the nongrowing areas
of the SPH (2, 13). Assuming that 12.5% of the
cAMP in a 24-mm SPH is present in the 3-mm
TABLE 2. Relative amounts of cAMP and cGMP
after a step up in light intensity from dark to 0.13
W/m2 at time zero'
Relative amt ± SD
Time (min)
cAMP cGMP
0 1.00 ± 0.05 1.00 ± 0.05
0.5 1.17 ± 0.18 1.16 ± 0.02
0.75 1.18 t 0.11 1.28 t 0.33
5.0 1.14 ± 0.15 1.08 ± 0.10
a Absolute amounts of cAMP and cGMP initially
present were 3,000 and 350 pmol/g (dry weight), re-
spectively.
556 NOTES J. BACTERIOL.
VOL. 153, 1983
growing zone and that the concentration of
cAMP in the growing zone decreases by 60%
after illumination, we should observe a 7.5%
decrease in the cAMP concentration of a 24-mm
SPH. According to the data in Table 2, 0.5 min
after illumination there was a 17% increase in
the mean cAMP concentration with an 18%
standard deviation of the mean. At 0.75 min,
there was an 18% increase and an 11% standard
deviation of the mean. A 7.5% decrease is 1.4 to
2.3 standard deviations away, and the chance of
our being off by this much is small, less than
10%. (It should be noted that the measurements
made by Cohen [2, 3], in which a 60% decrease
in the cAMP content was reported, were made
not with growing zones but with whole SPHs.)
There also was an increase in the mean cGMP
concentration as shown in Table 2. Again, the
differences may not be statistically significant.
Our evidence suggests that neither cAMP nor
cGMP are involved in light transduction in P.
blakesleeanus.
We thank H. C. Berg for helpful discussions, suggestions,
and critical reading of the manuscript; Teresita Legaspi for
technical assistance; and M. Delbruck for support.
This work was supported by National Science Foundation
grant PCM80-03846 to M. Delbruck.
LITERATURE CITED
1. Bergman, K., A. P. Eslava, and E. Cerda-Olmedo. 1973.
Mutants of Phycomyces with abnormal phototropism.
Mol. Gen. Genet. 123:1-16.
2. Cohen, R. J. 1974. Cyclic AMP levels in Phycomyces
during a response to light. Nature (London) 251:144-146.
3. Cohen, R. J. 1978. Aberrant cyclic nucleotide regulation
in a behavioral mutant of Phycomyces blakesleeanus.
Plant Sci. Lett. 13:315-319.
4. Delbruck, M., and W. Shropshire. 1960. Lectin and trans-
mission spectra of Phycomyces. Plant Physiol. 35:194-
204.
5. Delbruck, M., A. Katzir, and D. Presti. 1976. Responses of
Phycomyces indicating optical excitation of the lowest
triplet state of riboflavin. Proc. Natl. Acad. Sci. U.S.A.
73:1969-1973.
6. Jayaram, M., D. Presti, and M. Delbruck. 1979. Light-
induced carotene synthesis in Phycomyces. Exp. Mycol.
3:42-52.
7. Lipson, E. D. 1975. White noise analysis of Phycomyces
light growth response system. I. Normal intensity range.
Biophys. J. 15:989-1011.
8. Lipson, E. D. 1975. White noise analysis of Phycomyces
light growth response system. II. Extended intensity
ranges. Biophys. J. 15:1013-1031.
9. Lipson, E. D. 1975. White noise analysis of Phycomyces
light growth response system. III. Photomutants.
Biophys. J. 15:1033-1045.
10. Otto, M. K., M. Jayaram, R. M. Hamilton, and M.
Delbruck. 1981. Replacement of riboflavin by an analog in
the blue-light photoreceptor of Phycomyces. Proc. NatI.
Acad. Sci. U.S.A. 78:266-269.
11. Russo, V. E. A. 1977. The role of blue light in synchroniza-
tion of growth and inhibition of differentiation of stage I
sporangiophore of Phycomyces blakesleeanus. Plant Sci.
Lett. 10:373-380.
12. Steiner, A. L., D. M. Kipnis, R. Utiger, and C. Parker.
1969. Radioimmunoassay for the measurement of adeno-
sine 3',5'-cyclic phosphate. Proc. NatI. Acad. Sci. U.S.A.
64:367-373.
13. Tu, J. C., and S. K. Malhotra. 1973. Histochemical
localization of adenyl cyclase in the fungus Phycomyces
blakesleeanus. J. Histochem. Cytochem. 21:1041-1046.
NOTES 557


Absence of Significant Light-Induced Changes in cAMP Levels in Sporangiophores of Phycomyces blakesleeanus

Aberrant cyclic nucleotide regulation in a behavioral mutant of Phycomyces blakesleeanus.

Cyclic AMP levels in Phycomyces during a response to light.

Histochemical localization of adenyl cyclase in the fungus Phycomyces blakesleeanus.

Lectin and transmission spectra of Phycomyces.

Lightinduced carotene synthesis in Phycomyces.

Mutants of Phycomyces with abnormal phototropism.

Radioimmunoassay for the measurement of adenosine 3',5'-cyclic phosphate.

Replacement of riboflavin by an analog in the blue-light photoreceptor of Phycomyces.

Responses of Phycomyces indicating optical excitation of the lowest triplet state of riboflavin.

The role of blue light in synchronization of growth and inhibition of differentiation of stage I sporangiophore of Phycomyces blakesleeanus.

White noise analysis of Phycomyces light growth response system.

White noise analysis of Phycomyces light growth response system. I. Normal intensity range.

White noise analysis of Phycomyces light growth response system. II. Extended intensity ranges.

http://authors.library.caltech.edu/5921/1/LEUjbact83.pdf

Absence of Significant Light-Induced Changes in cAMP Levels in Sporangiophores of Phycomyces blakesleeanus

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main