Control of cell division in the filamentous green alga Zygnema
Item Type text; Thesis-Reproduction (electronic)
Authors Staker, Robert Dale, 1945-
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/318132 CONTROL OF CELL DIVISION IN THE
FILAMENTOUS, GREEN ALGA
ZYGNEMA
by
Robert Dale Staker
A Thesis Submitted to the Faculty of the
DEPARTMENT OF BIOLOGICAL SCIENCES
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE WITH A MAJOR IN BOTANY
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 7 S STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfill ment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library,
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
Robert W. Hoshaw Professor of Biological Sciences ACKNOWLEDGMENTS
The author wishes t:o express special! appreciation to Dr» Robert W » Hoshaw for.his many suggestions and guidance durihg the research of the problem and the writing of this manuscript<,
The author wishes to extend his gratitude to
Dr0 Robert Mellor for his criticism and suggestions»
A special thanks is extended to Dr» Willard
Van Asdall for his time in reading and criticizing this thesiso
I am indebted to Dr„ James Rosowski for his suggestion of the subject matter and for his helpful suggestions and techniques throughout this: study 0
iii TABLE OF CONTENTS
•' Page
LIST OF ILLUSTRATIONS o , . . . , = 0 . „ . .0 o 0 = v
LIST- OF TABLES © o o o o o o o o o o o o.o o o o o ' vxi.
ABSTRACT O O O O O O O 0 O O O O O O 0 0 0 0 O 0 0 VLl J»
INTRODUCTION O O O O O O O O O O O. O O O 0 0 0 0 0 1
REVIEW OF LITERATURE « o . 0 » . a « > = «, » = 4
MATERIALS AND METHODS . ».•»; , , » o 0 • « » „ . . ' 9
Experimental Organism «, » ' , e 0- « « » • «• 9 Medium and Inoculation « , » „ . o a. oooo « 10 Growth Conditions , « = 6 , a o . o o » o o » 10 Harvesting and Fixation 0 # «. 0 o « a 0 ■ a -o « 12 Mordanting and Staining » , = « » « * , , v 13 Counting. Mitotic Divisions o Vo , ,'a 14 Stages of Cell Division Defined » o o o ? 0 e 14 Prophase o ©. o * o o o o o o .o * o o o o .15 Me tap ha s e a © © o » o .o o o o o o ©o © © 15 Anaphase © © © © o © ©' © © © © © © © © © .15 Telophase © ©.© © © © .© o © o o © © o © © 15 . Quantity of Cell Division © © © © © © © © © © 15
OBSERVATIONS AND RESULTS © © , © © © © © © , = © © 17
Optimum Temperature for Growth © © © © © © © ©. 17 Optimum Light Intensity for Growth © © © .© © © 17 12 s12 hr Light-Dark Cycle © © © © © « © © © © 20 Mitotic Times and Average Percentage © © . © © 20 4 i20 hr Light-Dark Cycle © © © © © © © © © © © 26 10:14 hr Light-Dark Cycle V © © © © © © © © © 26 Continuous Low Light © © © © © © © © © © © © © 26 An Attempt to Quantify Mitosis .© © © © © © © © 27
DISCUSSION A N D •CONCLUSIONS © © © © © © © © © © © © . . 31
SUMMARY © a © © © o © ©o© © © © © © © © © o © © © a 37 LITERATURE CITED © « © © © © © V © © ©. © © V V © © 41
iv LIST OF ILLUSTRATIONS
Figure - Page
1o Growth of Zygnema plotted as a function of temperatureo Five plates were grown at, each temperature 0 0 0 „ „ 0 0 0 6 0 # o : 13
2S Grovrth of Zygnema plotted as a function of light intensity „ 0 V 0 0 » 0 „ 0 V . 0 „ 19 3<> Histogram showing, the pattern of cell division in Zygnema sp8 when grown on a 12 $12 hr light-dark cycle, at 20 X 1 C with a light intensity of 400-500 ft-c for two weeks 0 „ 0 » , 0 0 0 0 0 0 22
40 i Analysis of Figo 3 with percent of each phase of mitosis during the 7 Pil to the 1 AM division period 0 0 » ;• , 0 0 „ . • ; 23
5V Pattern of cell division of Zvanema sp0 grown.on 12$12 light-dark cycle at 400- 500 ft-c for 2 weeks and then subjected to.a 4 $20 hr light-dark cycle for 5 days0 The temperature was 20 t 1 C 0 0 , e „ „ 0 0 ; 24
6v- Histogram.showing the pattern of cell division in Zygnema spa grown on a 10:14 hr light-dark cycle at 20 t 1 at a light intensity of 400-500 ft-c for
.2 weeks o @ o 0 0 » o @ 0 0 @ @ @ @ ® 0 0 0 & ■ 23 7» : The pattern of cell division found in Zygnema grown for 2 weeks under 9 stand ard conditions9 and then subjected to continuous low light intensity of 40 ft-c (24:0 hr light-dark cycle) for a period of 5 days »• . , .. . , »• . » e , . 29
80 , The relati bus hip found in Zygnema when grown for 2 weeks under 9 standard . conditions9 arid then inoculated on plates subjected to continuous darkness (0:24 hr light-dark cycle) for 5, days 0 e 0 „ 30
v L 1ST OF ILLUSTRATIONS-- - Continued
Figure Page
9 o Controlled groxsrth environment in which cultures of Zv^nema were grown 0 « , .« « 0 '«' 39
10 o Petri plates shox«7ing the growth; of Zyanema after 14 days of 12:12 hr light-dark cycle? 400-500 ft-c and 20 i1 C o „ , , o 0 0 0 39
11 o Diagram showing the .Zvanema cell in prophase , stage of cell division. Nuclear membrane . has. dissolved and chromatin material has thickened .o o o o o » o e o oo^o o o o.o o o 40
12o Diagram of the metaphase cell in Zvahema ■ with chromosomes kt equator 0 0 0 , 0 «• * » 0 40
- 13 o Anaphase cell, in Zvanema with chromatids f migrating toward cell pole „ 0 , «; #• • o 0 « » . 40
14, Diagram of telophase cell with nuclear membrane reappearing, cytokinesis nearing completion * • , , , ., . .o .» . , , 40. LIST OF WBLES
Table
10 Composition of the gro^Tth medium used m this study @ o o o-o.o o o o o o o ©
2o' Stages of mitosis and their distributions0 Data from the 12 :12 hr light-dark. experiment using ’standard conditions’0
vii ABSTRACT
During the present investigation some of the
variables that "affect cell division in the greena filamen
tous alga ZyRnema were studied0 This alga was grown on
a defined medium in axenic culture under a controlled
environmento Various parameters that influence the
growth of Z y g n e m a and to an extent mitotic division^ were controlled to determine some of the optimum growth
requirements of this alga. An attempt;was made to determine the minimum amount of light required to synchronize cell division. Also it was confirmed that cell division occurs during the dark cycle of a diurnal light-dark cycle„ with most pf the cells dividing during the first 5 or 6 hr after the onset of darkness, A study of mitotic times was made with methods used by other workers, Since no cell division was found when Zygnema was grown under continuous low light intensity conditions s it is concluded that cell division in this genus is exogenously or environmentally controlled.
viii INTRODUCTION
Rhythmic processes of many organisms appear to
be under the control: of an "unacquired character/' some
times called a biological clock, Zeitgeber, or cue, that
is synchronized with some external environmental factor0
Rhythmic activities in organisms are sometimes referred
to as endogenous if they occur independently of environ
mental influences and exogenous if they occur as a direct
result of environmental influences (Sweeney and Hastings„
1962)o A classical example of an endogenous rhythm is
that of luminescence in Gonyaulax, a unicellular marine
, dinoflagellate (Sweeney and Hastings» 1958*)V'
■ Among the grpen algae which have been investigated
for 24-hr rhythms of mitosis, the following are reported to
exhibit peaks in the dark portion of the light-dark cycles
Closteriunu Cosmarlum, Hvdrodictvonfl UlothrixV Staurdstrum, ' ' ’ " ' ' 1 MouReotia «■■■, SpiroRvra; and Zygnema (Leedale, 1959) 0 The
rhythm was reported to be exogenous in these genera, ,
mitosis occurring at any time of the clock merely by adjust
ing the time of the dark period in a growth cabinet» How
ever, Leedale has presented no data on which this statement
is based»
Evidence that at least one of these algae, Zy&nema,
does not show an endogenous circadian rhythm lies in the
■: ' . • ■ i : - : • ■ . 2 fact that cell division in this alga does not occur in
continuous darkp as is the case with luminescence in
Gonyaulaxo Although cell division occurs in Zygnema
during the dark portion of the 12 s12 hr light-dark cycle*
it is thought to be a light-dependent event (Rosowski p
1969)0
Most researchers in this field have found that
the mitotic peaks occur during the dark period of the
circadian light-dark cycle» but certain algae under special
conditions show a peak in the light period0 Among these
algae are Stigeoclonium and Draparnaldia (Brandham and
Godwardp 1965), However, in the case of these two algaep
the investigators show background mitosis (about !%)»
indicating a certain degree of asynchrony or, as it is
. also called» logarithmicgrowths It appears that state
ments concerning the time at which division peaks occur
within the light-dark cycle, are more important than
statements about the time that the first mitosis begins0
In Zygnema grown on a 12 si2 hr light-dark cycle, it has
been shown that cell division occurs during the first 5 hr
of the dark cycle and begins about 1 or 2 hr before the
end of the light cycle ( R o s o w s k i 1969)«
The present investigation is designed to add to
some of the ideas of workers who have been previously
mentioned and to investigate further ceil division in
Zygnema when it is grown on a defined medium, under axenic conditions„ The objectives of the present inves tigation can be summarized as follows s
1o)to determine the temperature at which
v maximum cell division occurs in Zygnemao
2o) to determine the light intensity at which
maximum cell division in this organism
occurs o
30) to determine the minimum amount of light in
a 24-hr light-dark cycle needed to initate
synchrony of cell divisionD
4a) to determine whether cell division in Zygnema
is exogenously or endogenously controlled by
means of continuous low light conditions0 REVIEW OF LITERATURE
The genus Zygnema Is placed in the Division
Ghlorophyta because of its starch photosynthate (Smiths
1950)o Algae belonging to the order Zygnematales differ
from all other green algae in that there is an absence of
flagellated gamefes and fusion of gametes usually occurs
in conjugating tubes„ . The family Zyghemataceae probably has more species than any other family of filamentous green algae o A collection from ponds»_ ditches and slow moving streams are almost sure to show some species of this group, and there are over one hundred species of Zygnema widely distributed from sea level to the Alpine zone on all continents (Transeau, 1951)o
Zygnema was first described by. C0 A» Agardh, a
Swedish scientist in 1824« Morphologically Zygnema can be described as a simple, unbranched green filamentous alga, with cylindrical cells possessing a cell length slightly greater than the cell diameter» The protoplast has a central nucleus which is usually flanked by two stellate chloroplasts that lie axial to each other when
Considering the longitudinal axis of the cell0 However, more than t w o chloroplasts per cell can be observed in rare cases (Smith, 1950? Rosowski, 1969)0 Haptera also have been reported to occur in this genus„ Sexual reproduction is usually accomplished by, fusion of isogametes either in conjugation tubes or in the female gametangia 0
Akinetes are one way in which this genus survives during unfavorable conditions0 The identification of species within the genus is partly based upon vegetative dimen= sionso Also important is the characteristic of the mature zygospore wall (Transeau, 1951)0
It has been shown by various workers that events which occur in the life cycle of some: organisms can be synchronized with an environmental factorD Probably the most common method of Synchronization is by use of programmed light-dark regimes* One such event that is easily synchronized is that of cell division® Among the algaep cell division in Chlamydomonas moewusii has been . synchronized with the length of the light period (Bernstein,
1966)» This investigator found that cell division will occur with as little as 2-3 hr of light in a 24 hr period®
However£, in this alga multiplication with true regularity required a 4:20 hr light-dark cycle®
The problem of endogenous rhythms, or rhythms that occur as a result of some activity within the organism, has attracted the attention of. several investigators„
One of the best known investigations on the algae is that of the rhythm of luminescence exhibited by Gonyaulax polyedra (Sweeney and Hastings, 1958)» They reported that the rhythm persists under conditions of continuous low light intensity for a period of at least 14 days„ This
rhythm also remains a few days in the dark,period but is
lost after 4”6 days in continuous bright light. This
rhythm can be shifted to occur at any time of the day by
altering the timing of the light-dark period0
An investigation by Rosowski (1969) suggested
that the rhythm of cell division in the filamentous alga
Zygnema is an exogenous one. He showed that cells in
this alga» when grown in darkness (0 s24 hr light-dark
cycle)0 do not undergo cell division in the second 24 hr
period following the change from a 12s12 hr light-dark
cycle. However» he did not perform the crucial experiment
of monitoring cell division when cultures were grown at
.low light intensities and could not conclude positively
that the rhythm is exogenous,
The relative duration of each stage of mitosis
in the Desmidiaceae, another family in the Zygnematales„
was investigated by Brandham and Godward (1965), These
investigators were able to synchronize cultures of
Closterium siliqua on a 12:12 hr light-dark cycle, with
the lights on from 6 AM to 6 PM, In this work, the
percentage of mitosis was plotted as a function of time
and their graph shows one peak of cell division occurring
per day, and this peak occurs mostly in the first 6-7 hr
of the dark period. No background mitosis, was observed,
From this peak a second graph was made showing the percent of?each phase of mitosis as a function of time.
In this graph it was noted that the prophase curve from
5 s30 AM to 7 s30 AM is about parallel to the telophase curve from 6 8 30 AM to 8 s30 AMo Brandham and Godward infer from this observation that at equivalent points on the vertical axis of the plot nuclei are entering and leaving prophase at the same rate as they are entering and leaving telophase and that these nuclei are probably the same nuclei» These authors reason further that these points on the prophase and telophase curves represent cells at every stage of prophase and telophase and can be said to represent the mid-point of the stages„ They believe, therefore, that the horizontal distance between the two parallel sections of their prophase and telophase curves is actually the time required for nuclei to pass from mid-prophase to mid-telophase= They calculated this time to be 56 min in the case of C, sillqua0 Brandham and Godward concluded from their work that the overall rate of mitosis is dependent on the number of cells entering prophase and leaving telophase per unit time<,
.Mitotic time in the Desmidiaceae was also inves tigated by Waris (1950) who studied four species of
Micrasterias a The data obtained by Waris agree well with those of Brandham and Godward„ There is some discrepancy in length of prophase and telophase,.probably due to differences in individual definitions of what constitutes prophase and telophaseo
The influence of certain chemicals on cell division among the algae has been studied by various co authors, Tanliya (1963) has shown that sulfur in the form of a sulfate radical is required by some microalgae for cell division to occur0 He points out that cell division stops in the absence of this material„ Prasad and Godward
(1966) showed that gibberillic acid does not have the effect on Zygnema cruclatum that it does on sweet pea» regardless of the concentration, of the acid„ MATERIALS AND METHODS
Experimental Organism
The experimental organism used throughout this investigation was Zvanema sp„ (strain 922) which was obtained from the Culture Collection of Algae at Indiana
Universityo It was obtained in liquid culture and was not axeniCo This strain was isolated into axenlc culture by the potassium tellurite technique (Ducker and Willoughby0
1964)9 and maintained throughout this study in axenic cultureo Cultures were considered bacteria-free when filaments that were streaked on nutrient agar slants
(Difco) did not produce bacterial or fungal colonies. The choice of this particular algal strain was based on the research of Dr, James R, Rosowski„ who noticed that this particular strain did not undergo cell division when subjected to a continuous darkness condition. It was hypothesized that this strain of Zygnema exhibited an exogenous' rhythm with respect to cell division. This particular strain also will readily produce zygospores on a modified Bristols s solutions called. Godward * s C medium and hence may prove to be interesting as an organism for future study on conjugation. . 10
Medium ancl Inoculation
The medium used in this investigation was a modified
Bold0s medium described by Gauch (1966)„ Table 1 shows the composition of this:.medium., It was found that filaments will grow in the liquid medium, but more rapid growth occurs on a r/o agarized medium0 Also less space is required to store culture plates than with conventional bottle cultures 0
The inoculation procedure was similar to the one described by Rosowski (1969), and was carried out in a ster ile transfer room in order to maintain axenic conditions„
In preparation for the inoculation procedure a "razor bank" was made which consisted of six safety razor blades taped together, and was used to chop filaments» The chopping lasted for 5 min and was one method of randomizing cells9
The chopped filaments,were, then placed In a beaker with liquid growth medium and swirled to get an even distribu tion of the alga throughout the beaker. Four ml aliquots of the inoculum were sprayed on the agarized modified Bold's medium in plastic petri dishes. As far as is known, all cultures used in these experiments were axenic as indicated by the nutrient agar technique. Figure 10 shows the appear ance of growth after 14 days.
Growth Conditions
The experimental cultures were maintained under controlled environmental conditions, Two types of growth 11
Table 1„ Composition of the growth medium used in this studyoa
A modified Bristol,s basal medium (adapted from Gauchp 1966)„
Compound Concentration Percentage 1
CO 250 mg 0,025%
CaCl2 -’2H20 25 mg 0,0025%
■MgS04-7H20 7 5 mg 0,0075%
k2hpo4 75 mg 0,0075%
kh2po4 175 mg 0,0175%
NaCl 25 mg 0,0025%
EDTAb 50 mg 0,0050%
FeS04 »7H20 5 mg 0,00050%
H3BO3 11 mg 0 ,0011%
ZnS04 »I-I20 5,5 mg 0,00055%
MnCl2 o4H20 1 ,5 mg 0.00015%
CuS04 »5H20 1 0 5 mg 0,00015%
Co(N03)2 s6H20 0 .5 mg 0.00005%
aFigures cited here are. those used to prepare one liter of medium, with the volume brought up to one liter with deionized water» Agar was added at a concentration of 1%.
k Ethyl end iSmine tetraacetate-..'(disodium) was used as a chelating agent. chambers were used, a model 6610 growth cabinet manufac tured by the National Appliance Company of Portland, Ore gon, and a Percfval Model 1-36 L,growth chamber0 The light- dark period was programmed for a 12:12 hr light^dark cycle, with the lights on from 7 $00 AM to 7 s00 PM, except in the experiments with varying light cycles <, In order to maintain uniform light intensity and to assure proper ait circula tion, the shelves in the growth chamber were covered with aluminum foil with holes punched at regular intervals„ The light intensity was determined at the center of.each plate position by using a. Weston Illumination Meter Model 75®
The shelves in the growth chamber were leveled in order that the inoculum would be assured of uniform growth over the agar.surfaceo Also care was taken to have the plates poured and solidified on a level surface»
Originally the temperature was held at 20 C, the one exception being the temperature experiment in which temperature was the only variable» Taylor maximum- minimum thermometers were used to sample the temperature variation in the growth chambers, It was found that temperature would vary from shelf to shelf and. during the lightidark circadian cycle„ However, the temperature did not vary more than 20 1" 1 C» •
Harvesting and Fixation
After the algal material was subjected to test conditions it was harvested, Harvesting usually involved removing the algal mases from the plates using a safety
razor blade and placing the material into a vial which
contained about 20 ml of Jackson11 s fixative 0 Jackson1 s
fixative consists of a combination of methanol-ethanol~
chlorofora-acetone-proplonic acid in a 4:2:2:1:1
proportion (Waer, 1966) 0 Filaments were then cut with
a razor blade and centrifuged in a IEC Model Cl to top
speedc The supernatant lliiuid was removed and approxi
mately 2 ml of mordant added„ During the harvesting of
material from the temperature and light intensity
experiments» the following procedure, was usedo The
algal mass was scraped from the petri plates and placed
on pre-weighed watch glassesa The algal mass, on the
watch glass, was then allowed to dry in a Hydor Therme
Corps Model 615 D oven for 24 hr at 90 C 9 Next the watch
glasses were again weighed and the tare weight was subtracted
from the new weight, to give the values that appear in Fig„
1 and 2o
Mordanting and Staining -
The mordant was prepared in a similar manner to
that used by Rosowskl (1,969)» .Rusty nails were covered with concentrated propionic acid. The ferric propionate which resulted was filtered after 4 days and diluted to
50% with propionic acid.
After mordanting, the chopped filaments were
shaken for 1 min in order to randomize the sample. The 14 sample was in contact.with the mordant for about 5 min at
which time it was again centrifuged at top speed0 The mor
dant was decanted and a nuclear -stain*. propriocarmine, was
addedo .
The propriocarmine stain was prepared by boiling 500
ml of 50% propionic acid and adding 2,5 g of carmine„ About
2 ml of the stain was added to the filaments and placed in a
boiling bath for about 5 min. Again the sample was shaken
in order.to randomize it, A drop of the sample was removed
with a pipette, plated on a slide, covered with a number 1
- or 1% coverslip and the mount sealed with melted paraffin.
Counting; Mitotic Divisions
Cell counts were made using the oil immersion objec
tive of a Bausch and Lomb Research Laboratory: Microscope,
Counting was begun in the upper left corner of a slide and
all cells in a field of vision were counted. Vertical sweeps
toward the lower left corner of the slide were made, counting
all cells • in a field. At the lower left c o m e r the slide
was moved to the right until a new field was just in view and
then an upward sweep was made. The same procedure was used
. across the entire slide until 100,0 cells were .counted,
Stages of Cell Division Defined
At this time it may be beneficial to define briefly
the criteria used to distinguish the stages of mitosis in
Zygnema,. ... ■ -■■■■ 15 Prophase o During; the prophase stage there is a shortening and thickening of chromatin material„ Chromo some number counts were not made <. Also, during pro phase the nucleoli decrease in size and disappear;and the nuclear membrane disappears„
'Metaphase. The distinguishing feature of this phase of mitosis is that chromosomes migrate and align near the equatorial plane.
. Anaphase» Anaphase followed shortly:, with the dis tinguishing event being phat chromatids move toward opposite ends of the cell«
Telophase. In telophase the nuclear, membrane and - nucleolus reappeared. Completion of cell wall, formation and pyrenoid division is considered part of interphase.
After the pyrenoid divides, the nucleus migrates between the two daughter pyrenoids» This event is also considered to be part of interphase„
Quantity of Cell Division
Attempts were made to quantify the asmount of synchronization of cell division that occurred in Zygnema by means of a hemacytometer counting chamber and by use of a spectophotometer 20, Chopped filaments were placed in 40 ml of the growth, medium in two 125 ml Erlenmeyer flasks and covered with aluminium foil. One flask was . ■ ■ 16 placed in a grov/th cabinet with a 12 s 12 hr light-dark cycle at a temperature of 20 t 1 C and at a light ihtensity of
400 ft-Co The other flask wasSplaced in another growth chamber at 20 t 1 C at 400 ft-c under continuous light conditions o Harvesting was done, at hourly intervals for
48 hro It was assumed that growth curves under these conditions would be similar to those obtained by Cook
(1966) with stepwise curves resulting and cell division occurring synchronously in the dark period and logarith mic growth curves occurring in the non-synchronous condi tions. OBSERVATIONS AND RESULTS
Optimum Temperature for Growth
Attempts were made to find the optimum conditions
at which Zygnema will grow*. Two conditions were inves
tigated , temperature and light intensity«. In the
. temperature experimentg five different growth chambers
were employed, each chamber programmed for a different
temperatureThe light intensity was about 400 ft-c
with a 12s12 hr light-dark cycle„ Five petri plates
were inoculated and placed in each of the growth chambers.
The temperature was checked every other day with a .Taylor
maximum“minimum thermometer and the variance was t 1 C„
The cultures were allowed to grow for 7 days and then
harvested« Figure 1,shows the results of the temperature
experiment and 20 C is the optimum temperature for
obtaining maximum growth of Zygnema between 10-25 Co
Optimum Light Intensity for Growth
Another condition examined was that of light
intensityo Ten different light intensities were used
in four growth cabinets <, Five inoculated plates were
placed at each of the ten light intensities9 The
temperature was maintained at 20 C 1" 1 C and the cabinets
were programmed for 12s12 hr light-dark cycles„ The
''V ' ; ' ' - 17 Mean dry weight (mg/ plate) Figure 1. Growth of Zygnema plotted as a function of function a as plotted Zygnema of Growth 1. Figure 500 70 300 lOO
0 O temperature• eprtr. iepae eegona each at grown were plates Five temperature. 5 Temperat ure Temperat lO (C) 15 20 25
"a. *• cn o o Mean dry weight E 500 0 0 3 0 0 7 100 iue . rwh f ynm potd s fnto o lgt nest. this experiment are shown in Fig» 2 and optimum: light intensity was 400-500 ft-c. In.subsequent expefiments 20 C and 400-500 ft-c were used as . ’standard conditions“ for growing cultures„ 12i12 hr Llaht-Dark Cycle A preliminary experiment was performed to determine if cell division in Zygnema could be synchronized with the light-dark cycle„ In this experiment axenic filaments were inoculated oh agar plates containing the modified Bold's basal medium described in Table 1» The plates were subjected to 'standard conditions' and harvested at hourly intervals for 25 hr after 2 weeks of growth» The results are shown in Fig. 3. One thousand cells were examined at each hourly interval for stages of cell division. Mitotic Times and Average Percentage . Using the method of Brandham and Godward (1965) it was possible to calculate from Fig. 4 a time of about 62 min for nuclei to pass from mid-prophase to mid telophase. Assuming that mitotic time is unchanged over the harvesting periodj it is. possible to calculate mitotic times. Brandham and Godward pointed out that the. relative time required for a given nucleus to pass through any two stages of mitosis is directly proportional to fhe number of cells observed in each phase found in a sample of fixed material. 21 Out of the total of 7,000 cells examined during the actively dividing time, there were 340 cells in some stage of divisions(see Table 2).; From these 340 cells in some stage of division it was possible to obtain ratio (1)s (1) P 17.7: M 4,57: A 3*28: T 23.0. Since the time of 62 min takes into account only one-half of the prophases and one-half of the telophases, ratio (1) is modified as follows t (2) 8,85: M 4,57: A 3.28: %T 11.5. i'Then 62 min are divided up according to ratio (2) the following mitotic times are obtaineds (3) = 19,4; M = lOoO min; A = 7*21 min; = 25.3 min. Therefores Prophase =: 38,8 min Metaphase ~ 10,0 min Anaphase = 7,21 min - - Telophase '» 50 ,6 min This gives a total mitotic time of 107 min for Zyenema. The distribution of the various stages of mitosis under standard conditions over the entire division period was determined from the data shown in Fig, 4, The average percentage distribution of mitotic figures was as follows 5 Prophase 1,6%; Metaphase 0,3%; Anaphase 0,5% and Telophase 2,3%, From this it follows that the average percent of cells in division over the entire division period was 4,7% of the total cells observed. \ Percent Cell Division iue * itga soig h pten f el iiin n yrea p when sp, Zyrrnema in division cell of pattern the showing Histogram 3* Figure P - PM < 0 9:30 30 7 intensity of of intensity rw o a 21 h lgtdr cce t 0 1 wt a light a with C 1 - 20 at cycle light-dark hr 12:12 a on grown D a rk Peri od Peri rk a D I3 130 II.30 I I 400-300 ------:0 5:30 3:30 tc o to weeks. two for ft-c ie (hours) Time AM M- :0 :0 I 30 II 9:30 7:30 i h Period Light PM . 0 :0 5:30 3:30 1.30 ------ -- 7:30 in > Percent of each phase of mitosis A— 8 - A Figure 4. Analysis of Fig. 3 with percent of each phase each of percent 3with Fig. of Analysis 4. Figure - — -* ...... ------0.3 ■■■—■ A a \o t i se .s q h p L fic a me.t q. me.t pV\ 23 Percent Cell Division 4- s iue . atr o cl dvso o Zgea p gon n 21 lgtdr cycle light-dark 12:12 on grown op* Zygnema of division cell of Pattern 5. Figure PM — < '0 30 9 7'30 ak Period Dark at at yl fr dy. h tmeaue a 2 ± C. 1 ± 20 was temperature The days. 5 for cycle 400-500 30 -)<• tc o 2wesSdte sbetd o $0 r light-dark hr 4$20 a to subjected Sid"then weeks 2 for ft-c •30 3:30 AM 0 3 5 ie (hours) Time .jj. Light 7-30 Pe riod 9 30 30 4 <- ak Period Dark 1:30 — PM PM — 3 5:30 30 3 ----- 7.30 N> Dark Period Light Period I , , , T ime (hour s) Figure 6. Histogram showing the pattern of cell+division in Zygnema sp. grown on a 10:14 hr light-dark cycle at 20 - 1 C at a lignt intensity Ox 400-500 ft-c for 2 weeks. ■ 2 6 4:20 hr Light-Dark Cycle Axenic cultures of Zyanema-were exposed to ‘standard conditions' on a 12:12 hr light-dark cycleD Next the cultures were subjected to 5 days of a 4:20 hr . ‘ . ; . V . , ■ - \ light-dark cycle with a temperature of 20 "t 1 C and then harvested o- Figure 5 shows, the results of this experiment 0 It appears that no cell division is occurring by the fifth day of this treatment since no cells of the one .... ■ . ■ - ■; •e . .. thousand counted at each hr of the harvesting period showed# signs of cell division,, 10:14 hr Light-Dark Cycle After the results of the 4:20 and the 12:12 hr light-dark cycle experiments were known8 a 10:14 hr light- dark cycle„ The material was harvested and placed in Jackson’s fixative» Figure 6 shows the results of this experiment <, Complete, synchronization of cell division has been achieved and a 10:14 hr light-dark cycle may actually represent the proper light-dark cycle for synchronization to occur in this organism0 Continuous Low Light : Finally s, to verify the continuous low light inten- .sity experiment, a study was made using continuous darkness, Material was cultured under ’standard conditions’ for 2 weeks with the temperature held at 20 t 1 C, The material was then harvested and placed in a growth cabinet that was programmed for continuous darkness„ After 5 days of this treatment the cultures were harvested and v placed in Jackson’s fixative. The filaments were chopped and stained in, the usual manner and one thousand cells were examined for cell division* Figure 8 shows the results of this investigation* No cell division was found by the fifth day in continuous darkness* An Attempt to Quantify Mitosis Experiments were designed to quantify the amount of synchronization of cell division that occurred in Zygnema by means of a hemocytometer counting chamber and by use of a spectophotometer 20* However9 no statis tical differences were found in the hemocytometer counts or in the optical density readings as time progressed. This was probably due to the fact that growth of Zygnema is very slow in liquid culture* 28 Table 2„ Stages of mitosis and their distributions0 Data from the 12:12 hr light-dark experiment using ‘standard conditions 0’ — ------— ------— -- ---:----— ------— ------■ P M A T I 7 $30 14 1 2 2 981 8 $30 28 3 6 15 " 948 9 $30 26 11 6 35 922 Time 10$30 23 6 3 40 928 11 $30 8 5 2 37 948 12 $30 11 3 4 24 958 1 $30 4 3 8 . \ 985 totals 124 32 23 161 7,000 Percent Cell Division iue . h pten f el iiin on i Zgea rw fr ek under weeks 2 for grown Zygnema in found division cell of pattern The 7. Figure 3 x 4- 1 c 1 730 sity of 40 ft-c ft-c 40 of sity tnad odtos ad hn ujce t cniuu lwlgt inten light low continuous to subjected then and conditions* • standard PM IV. 0 3 (24:0 otnos o Lgt eid ' Period Light Low Continuous 3:30 Tm (hours) . Time hr ih-ak yl) o a eid f days. 5 of period a for cycle) light-dark AM 3:30 PM Percent Cell Division P - PM < Figure 8* The relationship found in Zygnena when grown for 2 weeks under under weeks 2 for grown when Zygnena in found relationship The 8* Figure 3 9 30 9 30 7 to tnad odtos ad hn ncltd n lts subjected plates on 1 inoculated then and conditions1 standard continuous darkness darkness continuous 11:30 Continuous 1.30 M A - :0 5:30 3:30 Time (hours) Dark ( 0;24 Period hr light-dark cycle) for for cycle) light-dark hr 7 30 I I '30 t 0 30 30 It K ----- PM - 5 3 5 30 5 .30 3 days. ------> — 7:30 7:30 DISCUSSION AND CONCLUSIONS Growth is sometimes thought of as being composed of several important events„ namely cell division, elongation, maturation and dlfferentiation<, This study involves the first of these parameters and some of the factors that control cell divis ion. Briefly, these factors include temperature, light intensity, and length of the light and dark periods when growth is on controlled cycles o It is believed that the results of this inves tigation are meaningful since Zygnema sp, (strain 922) was grown on a defined medium and in axenic culture„ Also some of the growth conditions were controlled by means of programmed growth chamberso Experiments were designed to learn about the growth patterns of this organism by finding optimum conditions in which this alga will grow, 'Standard conditions' were, defined and these included a temperature of 20 I- 1 C and 400-500 ft-c of illumination, since a maximum amount of Zygnema appears to grow optimally at this, temperature and this light intensity. This light intensity agrees well with that of Rosowski (1969)", Other members of the Zygnemataceae show varied temperature and light, intensity optima, Allen (1958) found that Spirogyra pratensls grows best at a light intensity . ■ . - ■ • . 32 below 5000 lux (about 460 ft-c)o She reported optimum growth on a 16 s8 hr light-dark cycle0 One strain of Sirogonium melahosporum shows optimum growth at 24-27 Cp :while Sp.lrogyra rectlsplra shows optimum growth near 16 C (Hoshaw* 1968)„ , The length of the light-dark cycle was varied somewhat in an attempt to find the minimum amount of light required to completely synchronize cell division in Zygnema„ It was found that Zygnema grown on a 12 ; 12 hr light-dark cycle shows some degree of asynchfony in the time of division* noticeably just prior to the onset of the dark cycleo Figure 3 shows this asynchrbny occurring at about 6:30 PM in the light cycle. Also it can be seen from the plot that cell division in this alga occurs only once during a 24 hr period and that the active period of division occurs in the last hr of the light cycle and the first 5 hr of the dark cycle on this particular diurnal cycle» It was hoped that some alternation of the 12.812 hr light-dark cycle could yield complete synchronization of cell division. Investigations .by Bernstein (1960* 1964* 19.66) indicated.that a 4:20 hr light-dark cycle is sufficent to induce cell division in Ghlamydomonas moewusii. This is the. minimum amount of light reported to date to induce cell division in the'algae. A similar situation was found by Senger (1965) to occur in Chlorella. For this reason a 4 $20 hr light-dark cycle was used in the present study, however, the results indicate that no cell division occurs in Zygnema when it id grown on this diurnal cycle. Although a 4:20 hr light-dark cycle may induce cell division in unicells such as Ghlamydomonas and Chiorella, it is believed that a 4:20 hr cycle is too short to induce cell division in Zygnema, After the results of the 4:20 hr light-dark cycle were knowns a 10:14 hr light-dark cycle was tried9 The results of this experiment showed that this is indeed the proper length of light duration required to induce cell division in Zygriema with no evidence of asynchrony =, It is believed that complete synchronization of cell division was found using this light-dark regime because of a lack of background mitosis r i 6 ea the percent, mitosis was zero at times other than the active dividing period (see Fig, 6), Also there were no peaks of cell division occurring in the light period as was the case in the 12:12 hr light-dark cycles However» it is not known whether the length of the dark period can vary with the same degree of synchronization occurring8 Perhaps a 10:13 hr light- dark cycle would work equally as wells No additional < experiments irivolying the length of the dark cycle were performed. An attempt was made to measure mitotic times as they occur in Zygnema when it is grown synchronously on .. / ■ V : 34 a 12 s12 hr light-dark cycle0 The procedure for obtaining . mitotic times was similar to the one used by Brandham and Godward (1965)0 These workers based their inferences about mitotic times on the fact that the slopes of the curves for prophase and telophase are essentially parallel and therefore the number of nuclei entering and leaving prophase is approximately equal to the number of nuclei entering and leaving telophase9 The slopes of the prophase and telophase curves of Fig, 4 are almost parallel and it is believed: that the same inferences and same method of determining mitotic times in Zvanema are valid. Although there are differences in the duration of the individual phases of mitosis, the overall time of cell division in Sirofeonium 707 (Wells, 1969), another member of the Zygnematales, is similar to Zygnema, The mototic time, as calculated by Wells for Sirbgonlum, is 107,4 min. The mitotic time calculated for Zygnema also agree fairly well with those obtained by Brandham and Godward (1965) for the desmid Closterium, which has a mitotic time of 98 min. Besides the two growth factors and the length of the light-dark cycle, experiments were designed to learn about the type of rhythm of cell division exhibited in Zygnema.when grown synchronously, Leedale (1958) has reported that cell division in Zygnema and various other green algae is exogenously controlled, Leedale, however, ' ' 35 shows no data to verify this statement0 Classical examples of endogenous rhythms are found in the diurnal leaf movements o f Phaeseolus multiflorus (Leinv/eber. 1956) and luminescence in Gonyaulax (Sweeney and Hastings, .1958)„ In both of these examples the critical experiment involved the use of a continuous low light intensity0 If indeed an endogenous rhythm is operating, it should continue in the presence of low light intensity= Also the rhythm should continue in continuous darkness for a period of timeo It has been reported that this rhythm persists under conditions of continuous low light for a duration of at least 14 days in the case of Gonyaulax„ This endogenous rhythm also remains a few days in the dark period but is Tost after 4=5 days in continuous bright . lighto The rhythm can be shifted to occur at any time of the day by altering the timing of the light-dark periods Sweeney and Hastings, have reported that optimum. . ■ - ■ ' ■ . ■ - growth of Gonvaulax occurs at a light intensity of about 1000 ft-c (T958)@ For their work with endogenous rhythms they used 100 ft-c for their continuous low light experiment, When one. applies this reasoning to Zygnema, a light intensity of 40-50 ft-c is obtained, since optimum growth for this organism is at about 400-500 ft-c0 In the experiment with continuous low light intensity in Zvanema, no evidence of cell division was found* It is suggested that cell division occurred only during one or more of the first four nights and by the fifth night or harvesting time,, cell division had ceased0 However„ an investigation of cell divisioh on the:first four nights was not performedo The same type of phenomenon was observed when continuous darkness was employed. It can readily be seen that this is different from the system which appears to operate in Gonyaulax0 It is therefore suggested that cell division in Zygnema is not endogen ously controlled but rather the environmental conditions are the controlling factors„ In other words, an exogenous rhythm seems to occur in the case of Zy,enema with respect to controlling the induction of cell division. SUMMARY , to A temperature of: 20 t 1 C was found to promote maximum cell division in Zygnema sp0 (strain 922), as indicated by increase in algal mass 0 2o A light intensity of about 400 ft-c promoted a maximum number of cell divisions in Zygnema, Both temperature and' light intensity are important to know when one is studying the growth of an organism since growth is sometimes, thought of as cell division, elongation, differentiation and maturation, 3v It was shown that Zygnema * like Sirogonlum«, has most of its cell division occurring during the first 5 hr of the dark period when grown synchronously on a 12:12 hr light-dark cyclea 40 The mitotic time was calculated in this alga to be 107 min and broken down as follows s Prophase - 3808 j Metaphase = 1000 min; Anaphase - 7.21r Telophase » 50 o6 min. These mitotic times compare well with those found in Sirogonium 707 and Closterlum. 5. Evidence indicates that cell division occurs only once in a 24 hr period in Zygnema grown on a 12; 12 hr and.a 10:14 hr light-dark cycle. • 37 There is complete synchronization of cell division in this organism when it is grown oh a 10:14 hr light- dark cycleo It has been shown that Zygnema grown on a 12:12 hr light-dark Cycle for 14 days and then subjected to continuous low light conditions for a period of 5 days shows no pattern of synchronization of cell division,, In fact, no evidence for cell division was foundo The same pattern is found when this • organism is grown under continuous dark conditions«V It is therefore believed that cell division in Zygnema is exogenously or environmentally controlled„ Figure 9, Controlled growth environment in which cultures of Zvschema were grown. Figure 10 a Petri olates showing the growth of Zygnema t after 14: days of 12 :12 hr light-dark cycle9 400-500 ft-c and 20 t 1 C, Figure 11 Diagram showing the Zygnema cell in prophase stage of cell division9 ■ Nuclear membrane has dissolved and chromatin material has thickened Figure 12 Diagram of the metaphase cell in Zygnenta with chromosomes at.equator® . Figure 13 Anaphase cell in Zygnema with chromatids migrating toward cell pole® Figure 14® Diagram of telophase cell with nuclear membrane reappearing9 cytokinesis nearing completiono 40 11 12 13 14 Cell Division in Zygnema LITERATURE CITED Allen, Mo A,, 1958. The biology of a species complex in Spirogyrao PhoD* Dissertation. Indiana Univ., Bloomingtone 240 p 0 Bernstein, E,, 1960» Synchronous division in cultures of Chi am yd omonas moewusii-. Science 131 :1528-1529 0 Bernstein, Eo, 1964, Physiology of an obligate photo autotroph (C^^ moeuxis 11) a Charact eristics of synchronously and randomly reproducing cells and an hypothesis to explain their popula tion curves* Jo Protbzoolo 11;56-746 Bernstein, E»,.1966o Physiology of an obligate photo- autotroph (Chiamvdomonas moewusii), II. The effect of light-dark cycles on cell division. Exp. Cell Res. 4 1 s307-315. Brandham, P. E. and M* B. E. Godward, 1965. Mitotic peaks and mitotic time in the Desmidiaceae. Arch. Mikrobiol. 51:393-398. . Cook, Jo R., 1966. Photosynthetic activity during the division cycle in synchronized -Eugleha gracilis. Plant Physiol. 4 1 s821-825. Ducker, S,.. C. and L. G. Willoughby, 1964. Potassium:: - tellurite as a bacteriostatic agent in isolating algae. Nature 202:210. Gauch, H ..G ., 1966. Studies on the life cycle and genetics of Zygnema» M.S. Thesis,. Cornell Univo 91 p. Hoshaw, R. W., 1968. Biology of the filamentous conju gating algae, p. 135 to 184, In D. F. Jackson (ede) Algae, man, and the environment. Syracuse Univ. Press. Leedale, G. F., 1959. Periodicity of mitosis and cell division in the Euglenlneae. Biol. Bull. 116: 162-174. 41 42 Leintvetier, F 0 J 9 , 1956, Ueber die Temperaturabhaengigkeit der Periodenlaenge bei der endogenen Tages- rhythmik von Phaseolus o Z, Boto 4 4 1337-364« Prasad» B» No and M. B0 EoGodward, 19660 Cytological studies in the genus Zygnema, Cytolbgia 3 1 s 375-391o Rosowski, Jo Ro j 19690 The pyrenoid of Zygnerfia: isolation and characterizationo Ph,Do Dissertation, Univ* Arizoj, Tucsono 138 p« Senger, H<. s 19658 . Teilungsinduzierende Wirkung des Lichtes auf ChlorelTa, Arch0 Mikrobiola 51 $307-322«, Smith, Go M o , 1950» The fresh-water algae of the United ; States, ^ :2nd editionMcGraw-Hill, New York. 719 p. • Sweeney, B. M. and J. Wo Hastings, 1958, Rhythmic cell division in populations of Gonyaulax polyedra, J , Protozool, 5i217“224. Sweeney, B. Mo and J, W, Hastings, 1962, Rhythms, p. 687 to 700, In Ro As -Lewin (ed,) Physiology and biochemistry of algae. Academic Press, New York, Tamiya, H,, 1963, Control of cell division in microalgae, Jo Cell Compe Physiol, 62 IIs157-174, Transeau, E. N,, 1951, The Zygnemataceae, -Ohio State Univ. Press o Columbus, 327 p. Waris, Ho, 1950, Cytophysiological studies on Micrasterias I,i nuclear and cell division. Physiol, Plantarium 3:1-16. Waer, R, D,, 1966, A cytological investigation of cell division in the filamentous green alga, Sirogonlum me1anosporum (Randhawa) Transeau, M,So Thesis, Univ, of Ariz,, Tucson. 59 p. Wells, C. Vo, 1969, Cytology of the green alga Slrogonium, Ph.D, Dissertation,' Univ, Ariz,, Tucson, 115 p.