EFFECT OF'CINNAMIC ACID ON THE GROWTH AND ULTRASTRUCTURE

OF CHLORELLA VULGARIS (BEIJERINCK)

by

LINDA LEE CHAMBERLIN, B.A.

A THESIS

IN

BOTANY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved _

Accepted

D e ^ of thfe praduati^ School

August, 1975

(- AC 805 T3

No.lU Cop. a.

^5T-<3/'~7r

ACKNOWLEDGMENTS

I am deeply indebted to Dr. Jerry D. Berlin for

his direction of this thesis.

11

CONTENTS

ACKNOWLEDGMENTS ii

Chapter

I. HISTORICAL REVIEW AND INTRODUCTION 1

II. MATERIALS AND METHODS 4

III. RESULTS 7

IV. DISCUSSION AND CONCLUSION 11

LITERATURE CITED 16

APPENDIX 19

• • •

111

CHAPTER I

HISTORICAL REVIEW AND INTRODUCTION

The premise that plants produced toxic substances

originated in 1832 when DeCandolle noted that some species

of plants appeared to be inhibitory to other species

(Bonner, 1950). Twenty years later, Liebig discounted

DeCandolle's hypothesis, and interest in the subject waned

until the early 1900's (Bonner, 1950) when Schreiner and

Reed (1908) first isolated toxic substances produced by

plants and identified the inhibitory chemicals as phenols.

Interest again lagged for many years because the phenomenon

was believed to occur in only a few instances in nature.

It was not until recently that the widespread nature of

these toxic substances was recognized. From the mid

1930's until the present, many cases of plant inhibition

have been studied and documented (Bonner, 1950; Whittaker

and Feeny, 1971; Pickett and Baskin, 1973).

Chemical characterization of these toxic compounds

showed them to arise from the amino acids tyrosine and

phenylalanine via the shikimic acid pathway (Neish, 1960;

Stafford, 1974). The combination of these aromatic

compounds with various other plant metabolites produced

a myriad of phenolic products. Of these products, cinnamic

acid is of interest due to its widespread distribution in

the plant kingdom. It has been found in higher plants such

as ferns, gymnosperms, and angiosperms (Glass, 1973). By

the processes of rainwash from leaves, root exudation,

decomposition of plant materials, and volatilization,

cinnamic acid derivitives accumulated in soils (Wang, Yang,

and Chuang, 1967). Cinnamic acid was shown to inhibit the

uptake of phosphate and potassium ions by barley roots

(Glass, 1973, 1974). Growth of Avena coleoptiles was

found to be inhibited by cinnamic acid (Marines and Hemberg,

1960). Dedonder and Van Sumere (1971) found that growth

of Chlorella vulgaris and Saccharomyces cerevisiae was

inhibited by cinnamic acid and some of its derivitives. In

a later study, they reported that cinnamic acid inhibited

14 uptake of C labelled phenylalanine (Van Sumere and

Dedonder, 1971). Low concentrations of cinnamic acid also

inhibited Paul's scarlet rose cell cultures (Danks, Fletcher

and Rice, 1975) and tobacco callus growth (Lee and Skoog,

1965).

At present, the mechanism of action of cinnamic acid

on these organisms is not known. Some studies point to

cinnamic acid as being an uncoupler of oxidative phos­

phorylation (Van Sumere, Cottenie, De Greef, and Kint,

1972; Stenlid and Saddik, 1962), while others have shown

that cinnamic acid is probably a weak uncoupler of oxidative

phosphorylation but the effect could not totally account

for the strong inhibitory effect observed (Tillberg, 1970).

In still another study. Glass and Dunlop (1974) demonstrated

that cinnamic acid caused a rapid, strong depolarization

of the membranes of barley roots, and they suggested that

this depolarization would account for inhibition of uptake

of inorganic ions. Sikka, Saxena, and Zweig (1973) found

14 that Chlorella pyrenoidosa which had incorporated C,

rapidly lost all labelled material after treatment with

quinone pesticides which were similar in structure to

plant phenols.

In spite of mounting evidence pointing to massive

membrane involvement, apparently no electron microscopic

studies have been conducted on living materials exposed

to cinnamic acid. Chlorella vulgaris was chosen as the

experimental organism for this study for several reasons.

First, Chlorella has been shown to be sensitive to cinnamic

acid (Dedonder and Van Sumere, 1971). Secondly, the use

of a unicellular plant disposed of the problems of cell to

cell transport and cellular location of the cinnamic acid

in the plant body. Finally, the glucosylation reaction

which acts as a detoxification process on introduced foreign

phenols in higher plants was absent in algae (Pridham, 1964)

CHAPTER II

MATERIALS AND METHODS

A culture of Chlorella vulgaris (Beijerinck) number 260

was obtained from the Algae Culture Collection at Indiana

University, Bloomington, Indiana. All cultures were grown

in 500 ml Erlenmeyer flasks containing 250 ml of sterile

modified Knop's solution with the following composition:

1.2 g KNO^, 2.5 g MgSO^-7 H2O, 1.1 g KH2PO-, 1 ml solution A

and 1 ml of solution B/1 (solution A contained 26.75 g

ferri ammoniumcitrate/1; solution B contained 1.32 g H^BO^,

2.05 g MnSO^-4 H2O, 0.10 g ZnCl2/ ^'^^ ^ CuCl2# 0.016 g

M0O-/I). The pH of the medium was 4.5 (Dedonder and

Van Sumere, 1971). Both stock and experimental cultures

were maintained at 25° C in an American Optical water bath

and filtered, prehumidified air was bubbled continuously

through all cultures. Overhead illumination was provided

by two 15 inch Gro-Lux fluorescent tubes (Sylvania) so that

light intensity at the glass surface was approximately

3000 lux. Cinnamic acid was obtained from Sigma Chemical -3

Company. Growth medium containing 10 M cinnamic acid

-4 was sterilized by filtration. Concentrations of 10 and

-5 -3 10 M cinnamic acid were prepared by diluting the 10 M stock solution with autoclaved modified Knop's solution.

Cultures for growth inhibition study were inoculated

with cells from an actively growing culture that was 7-10

days old to give a final concentration of 1- 1.5 x 10^

cells/ml of medium (Hase, Morimura, and Tamiya, 1957;

Zweig, Hitt, and McMahon, 1968; Van Sumere and Dedonder,

1971). Samples were withdrawn asceptically for analysis.

Growth of the culture was determined by two methods, first

using an Improved Neubauer hemacytometer for direct cell

counts, and second, by following optical density of the

culture on a Bausch and Lomb Spectronic 20 spectrophotometer

set at 640 nm.

Samples for electron microscopy were secured at time

intervals of 1 and 30 minutes and at 1, 12, 24, and 48

hours after incubation. These samples were centrifuged for

3 minutes at 1850 rpm in an International Clinical centrifuge

The supernatants were discarded and the cells were

resuspended in culture medium at pH 4.5 containing 5%

glutaraldehyde. The cells were fixed overnight at 4o C and

then post-fixed for one hour in 1% osmium tetroxide in 0.02 M

citrate buffer at pH 4.5 (Gomori, 1952). The samples were

dehydrated through a series of alcohols and propylene oxide

and embedded in Epon (Luft, 1961). Sections were cut on

an LKB Ultratome II microtome with a Du Pont dicimond knife

euid picked up on uncoated grids. The sections were stained

with uranyl acetate and post-stained with lead citrate

(Reynolds, 1963). The grids were viewed on an Hitachi

HS-8-2 electron microscope operating at 50 KV and an

Hitachi-llE-1 microscope operating at 75 KV.

CHAPTER III

RESULTS

Growth of the Chlorella was followed in two ways:

by cell counts done on a hemacytometer and by changes in

optical density of the culture (Figures 1 and 2). After

an initial lag period of 3 days, the control culture began

— 5 to grow rapidly. The culture in 10 M cinnamic acid also

lagged for 2 days and then began to grow rapidly. The

— 5 control culture and the culture with 10 M cinnamic acid

— 5 both seemed to grow at the same rates and 10 M cinnamic

acid did not inhibit the growth of the Chlorella. The

-4 culture containing 10 M cinnamic acid showed little change

prior to the fifth day post-inoculation. Five days after

inoculation both cell counts and optical density showed a -3

slight increase. The culture grown in 10 M cinnamic acid

showed virtually no change in cell number and the optical

density remained the same to the end of the experiment.

Twelve hours after inoculation, the culture containing -3

10 M cinncunic acid appeared bleached and had only a

vestige of green coloration. Within two days all traces

of green color were lost and the cells appeared white. -4 The culture with 10 M cinnamic acid began to lose

coloration after one day and faded gradually until only a

trace of green color was left by the third day after

8

inoculation. This culture never completely lost its faint _3

green color as did the culture exposed to 10 M cinnamic

acid. Neither the control culture nor the culture with

-5 10 M cinnamic acid lost any color and both remained

similar in color throughout all experiments.

Control cells exhibited normal ultrastructural

morphology (Figures 3 and 4). The cells had a large central

vacuole and the cytoplasm was distributed around the

periphery of the cell. Most cells had at least one

chloroplast usually containing a large pyrenoid surrounded

by starch plates. The chloroplasts were formed from

flattened stacks of thylakoid membranes and very often

starch grains could be seen accumulating within the

chloroplast (Figure 4). The nucleus was always adjacent

to the chloroplast and had dispersed electron dense areas.

The Golgi apparatus was located between the nucleus and the

plasma membrane (Figure 4). Small mitochondria with

tubular cristae were scattered in the cell.

Ultrastructural changes were not detected after

— 5 exposure of the Chlorella to 10 M cinnamic acid for 12

hours (Figures 5 and 6). The chloroplasts were normal in

appearance and starch was seen accumulating (Figure 5).

The nuclei were not changed nor were the mitochondria

(Figures 5 and 6). After two days of exposure to the

cinnamic acid, there were no observable differences between

-5 cells grown in 10 M cinnamic acid and the cells grown in

the control medium (Figures 7 and 8). The Chlorella grew

-5 well in 10 M cinnamic acid and cells which had finished

dividing and were bursting free from the mother cell wall

can be seen in Figure 7. Those cells were active photo-

synthetically as evidenced by the large accumulations of

starch in the daughter cells. In Figure 8, the chloroplast

has divided in preparation for cell division. Several

mitochondria were at the periphery of the cell and there's

a single nucleus.

After exposure for one minute to a concentration of

-4 10 M cinnamic acid, there were no apparent morphological

changes (Figure 9). The chloroplast and nucleus appeared

normal and the mitochondria were unaltered. A twelve hour

exposure to 10~ M cinnamic acid resulted in several

morphological types. Some cells appeared normal (Figure 10)

with no changes in the organelles, but other cells showed

severe damage (Figure 11). The plasma membrane was no longer

continuous and the thylakoid membranes were no longer

compact as in Figure 9 but appear to have spread out.

Membrane bound vesicles were seen at the periphery of the

cell. These vesicles were most likely plasma membrane

derived but could also have come from nuclear, chloroplast

or mitochondrial membranes. The nucleus and mitochondria

were no longer observable but may not have been in this

10

particular section. The pyrenoid and starch plates were

unchanged. A two day exposure to lO" M cinnamic acid

resulted in cells with disrupted thylakoid membranes.

Membrane fragments were seen in peripheral areas of the

cells (Figure 12). What appeared to be a degenerated

nucleus was found in the center of a cell, and the nuclear

envelope was no longer present.

No morphological changes occurred after one minute -3

exposure to a concentration of 10 M cinnamic acid

(Figure 13). The membranes were not affected and the

chloroplast appeared normal with a large pyrenoid body.

After one hour, the plasma membrane was fragmented and the

thylakoid membranes were spreading apart in the chloroplast.

Nuclei and mitochondria were not observed in these cells.

Again the pyrenoid body and the starch grains were not

altered. In other cells taken after one hour, short

membrane fragments were found (Figure 14). Large amounts

of starch were seen in these cells indicating that they

were photosynthetically active. By 12 hours, there was a

loss in the integrity of the thylakoid membranes of all

the cells and the only structures remaining without any

effects were the pyrenoid and the starch grains (Figure 15).

Short membrane fragments were found throughout the cells.

In Figure 16, membrane fragments were seen next to the

starch grains and in the pyrenoid. These fragments were

probably derived from the thylakoid membranes.

CHAPTER IV

DISCUSSION AND CONCLUSION

In the growth experiments, the lO""̂ M concentration of

cinnamic acid appeared to be lethal. Cell numbers and

optical density of the culture did not change after initial

inoculation. The culture turned white after 12 hours and

this phenomenon was consistently observed in all experiments. _3

After one minute, the cells in the 10 M cinnamic acid

culture revealed no morphological changes detected by

electron microscopy, but after one hour, the cells were

badly damaged. This damage was manifested by vesiculated

and fragmented plasma membranes while the delimiting outer

chloroplast membrane was disrupted. The inner thylakoid

membranes of the chloroplast appeared intact and similar

to the control cultures in ultrastructure. After 12 hours -3

of culture in 10 M cinnamic acid, the thylakoid membranes

had degenerated and, short lengths of a trilaminar structure

were seen in the cells. These fragments presumably were

membrane remnants most probably derived from disrupted

plasma, chloroplast, mitochondrial, or nuclear membrane

systems.

The loss of green pigmentation of the culture coincided

with the loss of integrity of the thylakoid membranes. In

studies involving photooxidation or bleaching of Chlorella

11

12

(Sironval and Kandler, 1958; Kandler and Sironval, 1959),

it was found that an induction period preceeded the photo-

oxidation of chlorophyll. These investigators suspected

that proteins which stabilized the chlorophyll molecules

were altered and this alteration led to a photooxidation

effect. In this study, the bleaching of cells exposed to

cinnamic acid was probably due to photooxidation of the

chlorophyll in the thylakoid membranes. Cinnamic acid

destroyed the thylakoid membranes where chlorophyll was

located and subsequently this membrane disruption led to

breakdown of the chlorophyll. The rate of chlorophyll

destruction was dependent upon the concentration of

cinnaonic acid. Bleaching occurred rapidly in those cells _3

that were exposed to 10 M cinnamic acid while those in

-4 10 M cinnamic acid bleached more slowly. A concentration

of 10 M had no bleaching effect. Studies involving

exposure of Chlorella pyrenoidosa to different quinone

pesticides resulted in a similar bleaching effect that

was concentration dependent with weaker concentrations

of the pesticides requiring a longer time for bleaching

of the chlorophyll to occur (Zweig, et al, 1968).

Despite the fact that cinnamic acid stimulates

respiration (Tillberg, 1970; Dedonder and Van Sumere, 1971),

-3 many starch grains were evident in cells treated with 10

M cinnamic acid. This would indicate that cell destruction

13

occurred so rapidly that even with increased respiration

rates, the starch was not broken down. Cultures treated

-4 with 10 M cinnamic acid had very little or no starch

remaining in their cells.

There were no apparent ultrastructural changes in

cultures treated with 10 M cinnamic acid until after 12

hours of culture. The treated algal cells displayed a

spectrum of effects, ranging from cells similar in

appearance to untreated cells to cells which were obviously

damaged. One explanation for this variation in degree of

-4 damage could have been that cells in the culture with 10

M cinnamic acid were of different ages and in different

stages of their division cycle. A second explanation was

that some cells had a detoxification mechanism which could

-4 have coped with a concentration of 10 M cinnamic acid.

Vance and Smith (1969) found that some strains of Chlorella

were resistant to the effects of some chlorophenoxyacetic

herbicides but the mechanism of resistance was not known.

Possibly the same type of resistance was operating in this

study. By the sixth day after inoculation, there was a

slight increase in both cell numbers and optical density

of the culture which indicated that some cells could have

survived the treatment and subsequently undergone division.

The number of cells which were able to survive was

extremely small as most cells after 2 days of exposure to

14

-4

10 M cinnamic acid displayed disintegration of chloroplasts

and other organelles were no longer recognizable.

Cultures exposed to 10~ M cinnamic acid appeared

similar to the control, and the nuclei, chloroplasts and

other organelles did not appear to be affected by this

concentration of cinnamic acid. Van Sumere and Dedonder — 5 (1971) had previously found that 10 M cinnamic acid had

an inhibitory effect on the strain of Chlorella vulgaris

(Pirson strain). Danks et al (1975) found that this

concentration inhibited Paul's scarlet rose cells. The

strain of Chlorella (IU-260) used for these ultrastructural

studies was not inhibited by 10 M cinnamic acid.

Cinnamic acid severely altered the plasma membranes

of Chlorella as evidenced by the loss of plasma membrane -3 -4 continuity in the 10 and 10 M treated cells. Fragments

of membranes could be seen in those cells. Since the

fragments were found in all parts of the cells, they

probably originated from several membranes including

chloroplast, mitochondrial, and nuclear membranes. That

total membrane structure was not destroyed indicated that

membranes were disrupted only at certain points along

their length. Apparently all the membrane systems were

affected. Similar destructive effects were found when

2, 4-dinitrophenol was applied to Chlorella pyrenoidosa

Chick cultures; chloroplast lamellae showed extreme

15

separation and derangement (Bertagnolli and Nadakavukaren,

1970). Cinnamic acid and 2, 4-dinitrophenol have similar

chemical structures and both may act similarly on Chlorella

membranes.

Glass and Bohm (1971) suggested that phenolic compounds

were not taken up by a specific transport carrier but

entered barley roots by passive movement through plasma

membranes. According to Glass (1973) there was a correlation

between lipid solubility of some phenols and their inhibitory

properties. He further suggested that when such compounds

passed through the plasma membrane, they altered some

membrane component and thereby changed the permeability of

those membranes.

Evidence presented in this paper showed that membrane

damage resulted from exposure to cinnamic acid and could

have accounted for the inhibitory effects exerted by other

phenolic acids. Phenolic acids play a role in the phenomenon

of allelopathy whereby a species of plant can inhibit the

distribution of other species by releasing phenolic acids

to the environment. Membrane damage to roots would certainly

be an effective method to inhibit growth. A better under­

standing of the mechanisms of allelopathy could lead to

development of crop species which would have a competitive

advantages over certain weeds.

LITERATURE CITED

Bertagnolli, B. L. and M. J. Nadakavukaren. 1970. Effect of 2, 4-dinitrophenoxyacetic acid on the fine structure of Chlorella vulgaris Chick. J. Phycol. 6:98-100.

Bonner, J. 1950. The role of toxic substances in the interactions of higher plants. Bot. Rev. 16:51-65.

Danks, M. L., J. S. Fletcher, and E. L. Rice. 1975. Effects of phenolic inhibitors on growth and metabolism of glucose-UL--*-̂ C in Paul's scarlet rose cell-suspension cultures. Amer. J. Bot. 62:311-317.

Dedonder, A. and C. F. Van Sumere. 1971. The effect of phenolics and related compounds on the growth and respiration of Chlorella vulgaris. Z. Pflanzenphysiol. 65:70-80.

Glass, A. D. M. 1973. Influence of phenolic acids on ion uptake. I. Inhibition of phosphate uptake. Plant Physiol. 51:1037-1041.

Glass, A. D. M. 1974. Influence of phenolic acids upon ion uptake. III. Inhibition of potassium absorption. J. Exp. Bot. 25:1104-1113.

Glass, A. D. M. and B. A. Bohm. 1971. The uptake of simple phenols by barley roots. Planta. 100:93-105.

Glass, A. D. M. and J. Dunlop. 1974. Influence of phenolic acids on ion uptake. IV. Depolarization of membrane potentials. Plant Physiol. 54:855-858.

Gomori, G. 1958. Microscopic histochemistry. University of Chicago Press. Chicago, Illinois, p. 219.

Hase, E., Y. Morimura, and H. Tamiya. 1957. Some data on the growth physiology of chlorella studied by the technique of synchronous culture. Arch. Biochem. Biophys. 69:149-165.

Kandler, O. and C. Sironval. 1959. Photooxidation processes in normal green chlorella cells. II. Effects on metabolism. Biochim. Biophys. Acta 32:207-215.

Lee, T. T. and K. Skoog. 1965. Effects of substituted phenols on bud formation and growth of tobacco tissue culture. Physiol. Plant. 18:386-402.

16

17

Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414.

Marines, N. G. and T. Hemberg. 1960. Observations on a possible mechanism of the inhibitor-B complex. Physiol. Plant. 13:571-581.

Neish, A. C. 1960. Biosynthetic pathways of aromatic compounds. Ann. Rev. Plant Physiol. 2:55-80.

Pickett, II, S. T. and J. M. Baskin. 1973. Allelopathy and its role in the ecology of higher plants. The Biologist (Phi* Sigma Soc). 55:49-73.

Pridham, J. B. 1964. The phenol glucosylation reaction in the plant kingdom. Phytochemistry 3:493-497.

Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208-212.

Schreiner, O. and H. S. Reed. 1908. The toxic action of certain organic plant constituents. Bot. Gaz. 45:73-102.

Sikka, H. C , J. Saxena, and G. Zweig. 1973. Alteration in cell permeability as a mechanism of action of certain quinone pesticides. Plant Physiol. 51:363-367.

Sironval, C. and O. Kandler. 1958. Photooxidation processes in normal green chlorella cells. I. The bleaching process. Biochim. Biophys. Acta 29:359-368.

Stafford, H. A. 1974. The metabolism of aromatic compounds. Ann. Rev. Plant Physiol. 25:459-486.

Stenlid, G. and K. Saddik. 1962. The effect of some growth regulators and uncoupling agents upon oxidative phosphorylation in mitochondria of cucumber hypocotyls. Physiol. Plant. 15:369-379.

Tillberg, J. 1970. Effects of abscisic acid, salicyclic acid and transcinnamic acid on phosphate uptake, ATP-level, and oxygen evolution in scenedesmus. Physiol. Plant. 23:647-653.

Vance, B. D. and D. L. Smith. 1969. Effects of five herbicides on three green algae. Tex. J. Sci. 20:329-337.

18

Van Sumere, C. F. and A. Dedonder. 1971. The effect of some naturally occurring and synthetic phenolics and related compounds on the uptake and incorporation of phenyla-lanine-l-14c by Chlorella vulgaris. Z. Pflanzenphysiol. 65:159-175.

Van Sumere, C. F., J. Cottenie, J. De Greef, and J. Kint. 1972. Biochemical studies in relation to the possible germination regulatory role of naturally occurring coumarin and phenolics. V. C. Runeckles and J. E. Watkin (ed.). Advances in Phytochemistry. Appleton-Crofts, New York. p. 165-221,

Wang, T. S. C., T. Yang, and T. Chuang. 1967. Soil phenolic acids as plant growth inhibitors. Soil Sci. 103:239-246.

Whittaker, R. H. and P. P. Feeny. 1971. Allelocheraics: chemical interactions between species. Science. 171: 757-770.

Zweig, G., J. E. Hitt and R. McMahon. 1968. Effect of certain quinones, diquat, and diuron on Chlorella pyrenoidosa Chick. (Emerson strain). Weed Sci. 16:69 73.

APPENDIX

19

20

m

I

Fig. 1. Graph showing log of the cell count per ml of culture for each cinnamic acid treatment. The counts were taken daily for one week.

21

p-K

—<D

- i n

— ^

CO

< o

liJ DC

- C O

111 CD <

—CM

'̂ cj o! h- K

o,d>cq K (O iq f^tOiD <o (6 (d

I I I I iO)00 h-

r I CO

( O CO

T CM

(D (6

- o

1W/lNnOO 1130 do 901

22

—3 Fig. 2. Graph showing the absorbance of 10 , 10 and 10"^ M cinnamic acid treated cultures at 640 nm. The readings were taken daily for one week.

-4

/r

30Nvauosav

24

Fig. 3. An electron micrograph of a control cell displaying normal morphology. Note the large central vacuole (V), nucleus (N) with electron dense areas, and chloroplast (C) containing a pyrenoid (P) with accompanying starch plates (SP). Small mitochondria (M) with poorly defined cristae are found in the cell periphery. 112,000X.

25

V̂

26

Fig. 4. A control cell showing large chloroplast (C) with starch grains. The Golgi apparatus (G) is located parallel to an elongated nucleus (N). 65,000X,

y < ^

27 I

28

3S

—5 Fig. 5. A cell which had been exposed to 10 cinnamic acid for 12 hours. The nucleus (N) and mitochondria (M) appear normal as does the pyrenoid (P) with starch plates (SP) and acciamulating starch grains (S). 45,000X.

M

/ ^

29

' ^ O

30

Fig. 6. A cell taken after 12 hours culture in lO" M cinnamic acid. Note the two chloroplasts and large central vacuole (V) with adjacent nucleus (N). Note also small mitochondria (M) with tubular cristae 44,000X.

/ ^

31

32

• I i '

— 5 Fig. 7. These cells were cultivated in 10/ M cinnamic acid for two days. Note large accumulations of starch (S) and an active Golgi complex (G) in the region of the nucleus (N). 31,000X.

33

34

l l i

II n

S R

t:

-5 Fig. 8. A cell exposed to 10 M cinnamic acid for two days. Note two large chloroplasts (C) with pyrenoids (P). A single nucleus (N) and many small mitochondria (M) are shown. 41,GOOX.

y^

35

/ . ^

8

36

i

m

Hj n\ SI 6!

M IIunit

Fig. 9. A Chlorella cell treated for one minute with 10~^ M cinnamic acid. Note single chloroplast (C) with stacked thylakoid membranes and pyrenoid (P). The nucleus (N) is large and an elongated mitochondrion (M) appears below the chloroplast. 108,000X.

*t^

I, 37

38

i

I 01

Fig. 10. A cell after a 12 hour exposure to 10 M cinnamic acid. The morphology appears normal in this cell with a chloroplast (C) and a large pyrenoid (P). The vacuole (V) is small in this view and small mitochondria (M) are in the peripheral areas. 49,000X.

-4

39

40

0^

Fig. 11. A cell also taken from a 12 hour culture containing 10"^ M cinnamic acid. Note that plasma membrane is discontinuous and thylakoid membranes (TM) in the chloroplast are spread apart. The pyrenoid (P) and starch plates (SP) appear normal. Small membrane-bound vesicles (VES) are found in the outer areas of the cell. 69,000X.

41

42

i

i I

«3I

Fig. 12. A cell after 12 hours exposure to 10 M cinnamic acid. Note membrane fragments (MF) and a degenerating nucleus (N). The pyrenoid (P) appears unaffected. 86,000X.

-4

y^

43

44

1!

i i

••Bi

-3 Fig. 13. A cell after one minute in 10 M

cinnamic acid. The ultrastructure appears unchanged There's a central vacuole (V) and a chloroplast (C) with pyrenoid (P) and starch plates (SP). 68,000X.

45

13

46

i g

I! 71-

-3 Fig. 14. Chlorella after treatment with 10 M

cinneunic acid for one hour. Note remaining starch grains (S) and small membrane fragments (MF). The cell wall is still intact. 39,000X.

47

48

-3 Fig. 15- After one hour in 10 M cinnamic acid,

the starch grains (S), starch plates (SP) and pyrenoid (P) are unaltered. Small membrane fragments (MF) are found through the cell. 46,00OX.

49

^^^ •^ \

• r r . ' ^ * r*

- \ V

, u •il

f

H

1 Fig. 16. A cell after 12 hours treatment with

lO" M cinnamic acid. Note many small membrane fragments (MF) found next to the starch grains (S) and within the pyrenoid (P). 67,000X.

51