RELATIONSHIP BETWEEN CHLOROPHYLL AND CHLOROPHYLLASE ACTWITY DURING Gazznme AND “GLUCOSE-BLEACHING" 0F CHLQRELLA Paomazcomas Thesis fax the flame of M; S. MCHIGAN SEAFE WWERSHY VECFQR GUELLEmO GANDZA _ - i974 - “Nb LI BRA R Y Michigan State University emoma av \‘1 "MB & SUNS' 800K BINDERY INL'. = LIBRARY BINDE ~; I 5 SPRIIGPORH'WI' " I ‘4 ti :1 L} n ._ C l ABSTRACT RELATIONSHIP BETWEEN CHLOROPHYLL AND CHLOROPHYLLASE ACTIVITY DURING GREENING AND "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES BY Victor Guillermo Ganoza It has been shown that chlorophyll synthesis or degradation in the alga Chlorella protothecoides can be induced by varying the nitrogen to glucose ratio in the culture medium. Chlorophyll synthesis was found to precede increase in chlorophyllase activity by 2-4 hours. During chlorophyll degradation chlorophyllase activity remained at high levels, suggesting a degradative role. Inhibitor experiments indicate that chlorophyllase is synthesized in the cytoplasm. Methyl pyropheophor- bide 2, a competitive inhibitor of the enzyme, showed no effect on chlorophyll synthesis or degradation but affected the chlorophyllase activity pattern in both cases. RELATIONSHIP BETWEEN CHLOROPHYLL AND CHLOROPHYLLASE ACTIVITY DURING GREENING AND "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES BY Victor Guillermo Ganoza A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1974 “U DEDICATION This thesis is dedicated to my parents Elena and Guillermo and to Marylee who gave me the support, encouragement and love necessary to make this degree a reality. ii ACKNOWLEDGMENTS I wish to express my sincerest appreciation to my academic advisor and committee chairman, Dr. Roger F. McFeeters. His aid and guidance were invaluable in the completion of this research. I would also like to thank Drs. Clifford L. Bedford and Richard W. Luecke, committee members. I am extremely grateful for the financial aid that I received throughout my master's program. My gratitude is extended to the Battle Creek Rotary Club and the Fullbright Commission of Peru for their aid. A special note of thanks to Dan M. Patrick for his graceful cooperation in typing the rough draft. iii TABLE OF CONTENTS Chapter Page I. INTRODUCTION . . . . . . . . . . . 1 II. LITERATURE REVIEW . . . . . . . . . 4 Chlorophyllase in Chlorophyll Synthesis . 5 Chlorophyllase as a Degradative Enzyme . 7 The Greening and Bleaching of Chlorella protothecoides . . . . . . . . 8 Effects of Antimetabolites on the Formation of Chlorophyll by Q. protothecoides . . . . . . . 10 Chloramphenicol . . . . . . . . 11 Cycloheximide. . . . . . . . . 12 III. MATERIALS AND METHODS . . . . . . . . 13 Culture and Propagation of Chlorella protothecoides . . . . . . . . 13 Bleaching Experiments . . . . . . . l5 Greening Experiments . . . . . . . 15 Cell Counts . . . . . . . . . . l6 Chlorophyll Content. . . . . . . . 16 Enzyme Determination . . . . . . . l7 Inhibition Experiments. . . . . . . 18 Chloramphenicol . . . . . . . . l9 Cycloheximide . . . . . . . . l9 Methyl Pyropneophorbide a. . . . . 19 IV. PESULTS O O O O O O. C O O O O O 22 Greening Under Normal Conditions . . . 22 Bleaching Under Normal Conditions . . . 25 Inhibitor Experiments . . . . . . . 32 V. DISCUSSION. . . . . . . . . . . . 60 Culture Maintenance. . . . . . . . 60 Data Presentation . . . . . . . . 61 iv 0"" Lil. Chapter Page Greening O O O O O O O O O O O 62 BleaChing. O O O O O O O O O O 63 Methyl pyropheophorbid a . . . . . 64 VI. SUMMARY AND CONCLUSIONS . . . . . . . 66 LITERATURE CITED . . . . . . . . . . . 68 LIST OF TABLES Table Page 1. Incorporation of methyl pyropheophorbide a into E. protothecoides . . . . . . . 58 vi Figure 1. LIST OF FIGURES Chlorophyll and chlorophyllase activity changes per liter of culture in green- ing cells of E. protothecoides . . . . Chlorophyll and chlorophyllase activity changes per 109 cells in a greening culture of C. protothecoides . . . . Chlorophyll and chlorophyllase activity changes per liter of culture in bleach- ing cells of C. protothecoides . . . . Chlorophyll and chlorophyllase activity changes per 109 cells in a bleaching culture of g. protothecoides . . . . Chlorophyll and chlorophyllase activity changes per liter of culture in greening cells of C. protothecoides with 5.3 x 10‘5M cycIohexImide added to the green- ing medium . . . . . . . . . . Chlorophyll and chlorophyllase activity changes per 109 cells in a greening culture of C. protothecoides with 5.3 x 10'5M cycloHeximide added to the green- ing medium. . . . . . . . . . . Chlorophyll and chlorophyllase activity changes per liter of culture in bleach- ing cells of C. protothecoides with 5.3 x 10‘5M Cycloheximide addéd to the bleaching medium. . . . . . . . . Chlorophyll and chlorophyllase activity changes per 109 cells in a bleaching culture of C. protothecoides with 5.3 x 10'5M cycloHexImide added to the bleaching medium. . . . . . . . . vii Page 24 27 29 31 34 37 39 41 Figure Page 9. Chlorophyll and chlorophyllase activity changes per liter of culture in green— ing cells of C. protothecoides with 2 x 10‘2M chlaramphenicol added to the greening medium . . . . . . . . . 43 10. Chlorophyll and chlorophyllase activity changes per liter of culture in bleach- ing cells of C. protothecoides with 2 x lO‘zM chlaramphenicol added to the bleaching medium. . . . . . . . . 46 ll. Chlorophyll and chlorophyllase activity changes per 109 cells in a bleaching culture of C. protothecoides with 2 x lO'ZM Chloramphenicol addedIto the bleaching medium. . . . . . . . . 48 12. Chlorophyll and chlorophyllase activity changes per liter of culture in greening cells of C. protothecoides with 500 nmoles of_methyl pyropheophorbide a per milliliter of culture medium — added . . . . . . . . . . . . 50 13. Chlorophyll and chlorophyllase activity changes per 109 cells in a greening culture of C. protothecoides with 500 nmoles 5f methyl pyropheophorbide a per milliliter of culture medium added . . . . . . . . . . . . 53 l4. Chlorophyll and chlorOphyllase activity changes per liter of culture in bleach- ing cells of C. protothecoides with 500 nmoles pe? milliliter of medium of methyl pyrophephorbide 3 added . . . 55 15. Chlorophyll and chlorophyllase activity changes per 109 cells in a bleaching culture of C. protothecoides with 500 nmoles of mEthyl pyropheophorbide a added per milliliter of culture medium . 57 viii C. 3‘ CHAPTER I INTRODUCTION Although chlorophyllase was discovered more than half a century ago little progress has been made in assessing its true role in chlorophyll metabolism. Many investigators have studied changes in chlorophyllase 'activity in relation to chlorophyll synthesis or degra- dation in different plants and lower organisms. Dif- ferences in techniques and results have failed to clarify the i2_zizg role of chlorophyllase. Hase and coworkers have carried out extensive investigation on the chlorOphyll metabolism of the alga Chlorella protothecoides (Aoki and Hase, 1964; Shihira- Ishikawa and Hase, 1964; Matsuka and Hase, 1969). Chloro- phyll synthesis and degradation in the alga are affected by the nitrogen to glucose ratio in the culture medium. By appropriately varying this ratio, it is possible to obtain cultures that synthesize or degrade chlorophyll. Hase's group, however, was concerned with the mechanisms that trigger the processes of synthesis or degradation rather than specifically with the degradation or synthesis Of chlorophyll. Chiba 33 31. (1967) studied the changes in chlorophyllase activity of the alga in relation to chlorophyll synthesis and degradation. They reported that an increase in chlorophyllase activity precedes the appearance of chlorophyll. Rapid synthesis of chlorOphyll occurred parallel to an increase in the activity of the enzyme. During chlorophyll degradation, a decrease in enzyme activity parallel to the rate of chlorophyll degradation was observed. Their results are expressed on the basis of a fixed packed cell volume. This causes some problems in reporting the data, espe- cially when cell division occurs during the course of the experiments. By combining the investigations of Base and coworkers with those of Chiba 35 31., much knowledge can be gained on the nature and role of the enzyme in the cell. Using metabolic inhibitors it is possible to eliminate synthesis of undesirable components that could mask the relationship between chlorophyll and chlorophyllase. More specific inhibitors, such as specific inhibitors of the enzyme, could provide definite information as to the true role of the enzyme in vivo. This study extends the work done by Chiba gt_§1. (1967). Changes in chlorophyll and chlorophyllase activity are reported on the basis of a liter of culture as well as on a fixed cell number. The per liter expression allows one to compare changes expressed on a constant base, while the fixed cell number expression allows comparison with Chiba's results. Metabolic inhibitors were added to cultures during synthesis and degradation of chlorophyll. Their effects on the pro- cesses of synthesis and degradation of chlorophyll, as well as their effect on chlorophyllase activity during these processes were studied. CHAPTER II LITERATURE REVIEW While trying to isolate crystals formed in leaf sections treated with alcohol, Willstatter and Stoll (1913) discovered chlorophyllase. Chlorophyllase (chlorophyll-chlorophyllido-hydrolase, EC 3.1.1.14) catalyzes in vitro the removal of the phytol group from chlorophyll a, chlorophyll 2, their respective pheo- phytins, and other phytol esters. Chlorophyllase is widely distributed in nature. It appears that it is present in all chlorophyll con- taining plants. An extensive study of its distribution in higher plants has been carried out by Mayer (1930). Chlorophyllase has also been found in gymnosperms and ferns (Willstatter and Stoll, 1913), in purple bacteria (Holden, 1963), and in several families of algae (Barret and Jeffrey, 1964). The enzyme occurs in all parts of the plant. However, stems, roots, and seeds have a much lower activity than leaves (Holden, 1963). Ardao and Ven- nesland (1960) suggest that chlorophyllase is localized (U DJ in a chlorophyll-lipoprotein complex. Stobart and Thomas (1968) indicated that chlorophyllase of Kalanchoé tissue cultures may be located in lipoprotein aggregates of the stroma. Chlorophyllase is insoluble in water. It is usually extracted from plant tissues by using detergents or suitable organic solvents (Holden, 1961; Shimizu and Tamaki, 1962). Ogura (1969) successfully extracted chlorophyllase of young tea leaves with water. However, as the leaves matured, chlorophyllase became more dif- ficult to extract. Since chlorophyllase substrates are also insoluble in water, the reaction has been commonly carried out in an aqueous acetone medium (Willstatter and Stoll, 1913). Klein and Vishniac (1961) devised a method that allows the reaction to proceed in an aqueous medium by using detergents to solubilize sub— strates. This procedure has been used by Stobart and Thomas (1968) and by McFeeters gt_§1. (1971). Chlorophyllase in Chlorophyll SynthesIs Although the reaction catalized by chlorophyllase £2.21EES normally favors pigment hydrolysis, a few investigators have successfully reversed it. Will— Stfitter and Stoll (1913), in their original description of chlorophyllase, reported synthesis of chlorophyll from chlorOphyllide and phytol using an air-dried meal of Heracleum leaves. Shimizu and Tamaki (1963) were able to synthesize chlorophyll from chlorophyllide and phytol and pheophytin from pheophorbide and phytol. Chiba gt_gl. (1967) synthesized chlorophyll from methyl chlorophyllide and phytol. Ellsworth (1971; 1972) reported synthesis of pheophytin both by direct esterif- ication of pheophorbide and by transesterification of methyl pheophorbide with phytol. Holden (1961) found that chlorophyllase activity increased when etiolated bean seedlings were exposed to the light and chlorophyll synthesis began. Shimizu and Tamaki (1963) followed seasonal changes in tobacco leaves and found that chlorophyllase activity paralleled chlorophyll synthesis and degradation. Chiba st 31. (1967) found similar results in cells of E. protothe- coides. Ellsworth and Aronoff (1968) and Aronoff gt 31. (1971; 1972) described a mutant of Chlorella which could not form chlorophyll, but instead formed chlorophyllide 3. Chlorophyllase activity in the mutant cells was 20 - 25% of that of the wild type. Reduction in the chlorophyll content of various fruits parallels decrease in chlorophyllase activity (Sudyina, 1963). Sudyina (1963) also studied chloro- phyllase activity in leaf sections of box elder (éEEE negundo L.) and showed that activity was highest in greening sections, somewhat lower in the dark green sections, and lowest in the albino parts. These results tend to link chlorophyllase with chlorophyll synthesis. Indirect support for a synthetic role of chloro- phyllase has been given by Park E£.E£' (1973). They found that phytol can be demonstrated in yellow leaves in quantities comparable with those of green leaves only after saponification. They assumed that the phytol ester compound in yellow leaves is a fragment of ring IV of the chlorophyll molecule. Chlorophyllase as a Degradative Enzyme Looney and Patterson (1967) have reported increased activity of chlorophyllase during the respir- ation climacteric of apples and bananas. Rhodes and Wooltorton (1967) reported identical findings in apples. During the climacteric chlorophyll degradation occurs. Ogura (1969) reports that chlorophyll of young tea leaves increases while chlorophyllase activity is decreasing. Ziegler and Schanderl (1969) found that chlorophyllase activity in a mutant of Chlorella increases while photobleaching. During dark bleaching chlorophyllase activity increased and pheophorbides accumulated. Bailiss (1970) has reported an increase in chlorophyllase activity and chlorophyll degradation in cucumber cotyledons infected with cucumber mosaic virus. Based on these and other similar observations, some authors suggest that the first step of chlorophyll degradation is hydrolysis of chlorophyll by chlorophyllase (Ziegler and Schanderl, 1969). The data in the literature do not give firm support for either a synthetic or degradative role for chlorophyllase. Certainly additional information on the relationship of chlorophyllase activity to chloro- phyll metabolism will be required before the biological role of chlorophyllase can be determined. The Greening and Bleaching of ChIorelIa protothecoides Shihira—Ishikawa and Hase (1964) found that pigmentation of cells of E. protothecoides depends upon the nitrogen/glucose (N/G) ratio of the culture medium. At high N/G ratios, green cells with fully developed chloroplasts are obtained. Low N/G ratios give bleached (white) cells with degenerated chloroplasts. At inter- mediate N/G ratios, light green to yellow cells may develop. Upon transferring to a new medium with appro- priate N/G ratio, cells of all colors are intercon- vertible. Fructose, galactose, glycerol, and acetate also cause chloroplast degeneration. This degeneration is enhanced by darkness and delayed by light (Shihira- Ishikawa and Base, 1964; Takashima gt 31., 1964; Aoki and Hase, 1965). Bleaching of the cells has been shown to be an aerobic process requiring oxidative phos- phorylation (Matsuka and Hase, 1965). This led them to suggest that an 0 depending step is needed for 2 chlorophyll degradation to occur in the living cells. Greening has been shown to be light dependent (Shihira-Ishikawa and Hase, 1964). However, "etiolated" cells which synthesize a small amount of chlorophyll in the dark are produced at appropriate N/G ratios (Aoki and Hase, 1964). Greening of these cells differs from greening of normal bleached cells in that no lag period in chlorophyll synthesis occurs upon exposure to light (Aoki and Base, 1964). From these results, Ochiai and Hase (1970) infer that the light effect is of two types. A long-term effect during which formation of G-amino levulinic acid is initiated, and a short-term effect during which chlorophyll is formed. The light induction of chlorophyll formation in bleached cells appears to be mediated by nonchlorophyllous photore- ceptors which are most sensitive to blue and yellow light (Sokawa and Hase, 1967). 10 Actinomycin, a DNA-directed RNA synthesis repressor, inhibits the light independent phase more strongly. Glucose represses RNA and protein synthesis during the light-dependent phase (Shihira-Ishikawa and Base, 1965). Aoki and Hase (1965) and Aoki, Matsubara, and Hase (1965) showed that no "new" cell formation was necessary for greening or bleaching of the cells but synthesis of nucleic acids and protein were essential for greening. Effects of Antimetabolites on the Formation of Chlorophyll by C. protothéCoides Chlorophyll synthesis of E. protothecoides has been inhibited by various antimetabolites. Actinomycin C, a suppressor of RNA synthesis, can inhibit chloroplast development in bleached cells if applied before provision of urea and light (Aoki and Hase, 1964). Mytomycin C, an inhibitor of cell division, has no effect on chloro- phyll synthesis or degradation (Aoki and Hase, 1965; Aoki, Matsubara, and Hase, 1965). Uncouplers or oxi- dative phosphorylation have been found to inhibit chlorophyll degradation in bleaching cells (Matsuka and Hase, 1965). Inhibition of greening in E. pggggf thecoides has also been shown by S-fluorouracil, acridine orange, dihydrostreptomycin (Aoki and Base, 1965), 11 Chloramphenicol (Aoki, Matsubara, and Hase, 1965; Aoki, Matsuka, and Base, 1965), and cycloheximide (Matsuka and Hase, 1968). Bleaching has been inhibited by cycloheximide. Partial inhibition of bleaching has been shown by Chloramphenicol, puromycin, and ethionine (Matsuka and Hase,_1968). Chloramphenicol Chloramphenicol has been found to inhibit protein synthesis of chloroplast ribosomes due to its preferential binding to this type of ribosome (Anderson and Smillie, 1960; Margulies and Brubaker, 1970). Margulies (1968) reported that a particular protein fraction, normally present in small amounts, increased after illumination of Chloramphenicol-treated leaves, but an overall inhibition of protein synthesis occurs. It appears that chlorophyll synthesis inhibition is a secondary effect of protein synthesis inhibition (Ben Shawl and Markus, 1969). Bleaching of g. protothecoides is not totally inhibited by Chloramphenicol (Aoki, Matsuka, and Base, 1965; Matsuka and Hase, 1968). However, greening of algal cells is at least partially inhibited (Aoki, Matsuka, and Hase, 1965; Czygan, 1966; Hoober and Siekevitz, 1968; Smith-Johansen and Gibbs, 1972). 12 Cycloheximide Cycloheximide has been shown to inhibit protein synthesis in the cytoplasmic ribosomes (Mahler et_al., 1968; Hoober and Siekevitz, 1968). Other effects of cycloheximide include inhibition of chloroplast DNA synthesis (Drilica and Knight, 1971), inhibition of chloroplast membrane formation in Chlamydomonas reinhardi (Hoober and Siekevitz, 1968), inhibition of cell division in Euglena (Bishop and Smillie, 1970), and nuclear DNA synthesis in g. pyrenoidosa (Wanka and Moors, 1970). Glucose assimilation by cells of E. protothecoides was inhibited and no bleaching observed if cycloheximide was added to the culture at the same time as glucose. If added later, bleaching continued for a short period before it stopped completely (Matsuka and Hase, 1969). CHAPTER III MATERIALS AND METHODS Culture and Propagation of Chlorella protothecoIdes Chlorella prothotecoides (ACC #25) was obtained from the University of Indiana algal culture collection (Bloomington, Indiana). The culture was maintained on a proteose agar slant (Starr, 1964) at 4°C. To propagate the culture, the agar slant was incubated with 5 m1 of the basal inorganic medium described by Shihira-Ishikawa and Hase (1964) contain- ing 0.5% urea and 1% glucose. The composition of a liter of medium is: KH2P04, 0.79; K HPO 0.3 g; 2 4' 4, 4o7H20, 3 mg; thiamine hydro- chloride 10 pg, Arnon's "A5" mineral solution, 1 m1. MgSO 0.1465 g; FeSO A liter of Arnon's "A5" mineral solution contains H3BO3, 3.86 g; MnC12-4H20, 1.18 g; ZnSO4~5H20, 0.222 g; CuSO4- 20, 0.079 g; M0203, 0.0187 g (Mitsuda 33 31., 1970). Glucose and urea are added as indicated to give greening SR or bleaching conditions (Shihira-Ishikawa and Hase, 1964). Phosphates were sterilized separately from the rest of the medium to avoid precipitation and cloudiness of 13 14 the medium. Thiamine hydrochloride was filtered through a sterilized 0.45 mm Millipore filter to avoid degra- dation by heat. The agar slant with 5 ml propagation medium was incubated at room temperature for 48 hours under con- tinuous room light. The cell suspension was then trans- ferred to a 500 ml erlenmeyer flask containing 200 ml of the propagation medium. The culture was placed in the dark and shaken on a reciprocal shaker at a rate of 110 strokes per minute. Temperature was kept con- stant at 24°C. After 48 hours, cells were transferred to a Fernbach flask containing 1.5 l of propagation medium. Incubation was continued in the dark under the same conditions. Aereation of the Fernbach flask was found to be necessary to obtain optimal growth. Air was pumped through a sterilized 0.45 mm Millipore filter with a Cenco Pressovac 4 Pump (Central Scientific Com- pany, Chicago, Illinois) and humidified by bubbling it through sterilized water. The air outlet of the Fern- bach flask was made out of glass tubing with three con- secutive "U" bends to avoid bacterial contamination. Aereation rate was approximately 60 ml/min. After 72 hours, cells were harvested by centrifuging (3-4 min., at 8000 x g) under sterile conditions. The cells, after two washings with sterilized distilled water, were 15 ready for subsequent transfers. Most cells at this time had a yellow color, but a few were yellow-green. Bleaching Experiments The yellow cells were transferred to basal medium containing 0.5% urea and grown in the light (900 lux at the culture surface) until a deep green color was obtained (chlorophyll content was then 0.15 mg per 109 cells or greater). Agitation, aereation and temperature conditions remained the same as earlier described. The green cells were harvested under sterile conditions, washed twice with sterile distilled water and transferred to basal medium containing 1% glucose. The culture was then kept in the dark. All other con- ditions remained constant. The set-up for the experiment required two Fernbach flasks linked in series with each containing 1.5 l of medium. Bleaching was initiated at the point where cells were transferred to the basal medium containing 1% glucose.- Samples were taken at appropriate intervals to determine dry weight, number of cells, chlorophyll content, and chlorophyllase activity. Greening Experiments The yellow cells obtained after the initial propagation period were harvested as earlier described. The cells were then transferred to basal medium 16 containing 1% glucose and incubated in the dark for 48 hours under standard conditions of aereation, agitation, and temperature. At the end of this period, the cells were harvested and suspended in basal medium containing 0.5% urea. Incubation was done under illumination (900 lux at the culture surface) with all other conditions remaining constant. Samples were taken at appropriate intervals and analyzed in the same way as for the bleaching experiments. Cell Counts Cells were counted by using an improved Levy- Housser counting chamber (C. A. Hausser and Son, Phila- delphia, Pennsylvania). Cell numbers are expressed as number of cells per milliliter. Chlorophyll Content Eighty milliliters of culture suspension were centrifuged and the supernatant discarded. The cells were then suspended in 1-2 ml of acetone. The suspension was filtered through a fritted glass funnel layered with 0.6 9 sea sand. The cells and sand were then transferred to a mortar and ground. The sea sand was used as an abrasive. The powder resulting from grinding of the cells was suspended in acetone and filtered through a fine fritted glass funnel. This procedure of grinding and filtering was repeated at least twice l7 and until no more chlorophyll could be extracted. All of the filtrates were combined, made up to 10 m1 and used to determine chlorophyll content. Readings of the extract were made at 664, 647, and 750 nm with a Beckman DU spectrophotometer equipped with a Gilford absorbance indicator. The 750 nm reading was subtracted from 664 and 647 measurements to correct for light scattering. Equations developed by Ziegler and Egle (1965) were used. 11.78 A - 2.29 A Chlorophyll 3 (mg/1) 664 647 - 4.77 A Chlorophyll 3 (mg/l) 20.05 A 647 664 These equations are for 80% acetone solutions. Since large quantities of lipid material would precipi- tate under these conditions, it was necessary to use 3.3. 100% acetone solutions and correct the absorbance reading for 80% solutions. The absorbance readings were divided by 1.04 (Ziegler and Egle, 1965). Results were expressed in mg per liter of culture and as mg per 109 cells depending on how the data were reported. Enzyme Determination The powder remaining from the chlorophyll extraction was further extracted for 24 hours in the cold with 3 ml of 0.012 M acetate - 0.06 M phosphate - 18 0.012 M borate buffer, pH 7.50 containing 0.5% Triton X-100. The powder was then separated by centrifugation at 27,000 x g for 10 min. Activity of the supernatant was measured by the procedure of McFeeters 33 31. (1971). The reaction is run in an aqueous medium (0.012 M acetate - 0.06 M phosphate - 0.012 M borate buffer pH 7.50, con- taining 0.2% Triton X-lOO). Pheophorbide 3'is separated from pheophytin 3|by shaking an aliquot of the reaction mixture in a 60:40 hexane - acetone solution with enough KOH to raise the pH of the aqueous phase to 8.5. The pheophorbide and the enzyme remain in the lower aqueous acetone phase while the pheophytin is transferred to the hexane phase. Absorbance readings of the lower phase were done at 750 and 667.5 nm (red absorption maximum for pheophorbide 3). Reactions were run for 2 hours. Samples were taken every 40 minutes. Results were expressed as nmoles of pheophytin 3 hydrolized per hour per liter of culture. In the case of the 109 expressions, results were expressed as nmoles of pheophytin 3 hydrolyzed per hour. Inhibition Experiments The effects of Chloramphenicol (Sigma Chemical Company), cycloheximide (Calbiochem), and methyl pyro- pheophorbide 3 on chlorophyll content and chlorophyllase activity of greening and bleaching cells were studied. 19 Chloramphenicol Green and white cells were incubated in the dark for 15-16 hours in the basal medium containing chlor- amphenicol (2 x lO-ZM). The green cells were kept in the dark and glucose was added (final concentration 1%) to the medium. The white cells were transferred to the light and urea was added to the medium (final concen- tration 0.5%). Control cultures without inhibitor, but otherwise treated in the same way, were carried out at the same time. Conditions of aereation, temperature and agitation were the same as described for bleaching and greening. Samples were taken at appropriate intervals after the addition of urea or glucose and analyzed for cell number, dry weight chlorophyll and chlorophyllase. Cycloheximide Cells were treated in the same way as with Chloramphenicol except that cycloheximide (5.3 x 10-5M) was used as the inhibitor. Methyl Pyropheophorbide a Both green and white cells were incubated in the dark for 24 hours. At the end of this period methyl pyropheophorbide 3, suspended in a 2% Triton X-100 solution, was added to the culture. 20 Methyl pyropheophorbide 3 dissolved in 80:20 ether-acetone and was suspended in a 2% Triton X-100 solution. The solvents were then evaporated at low pressure. The concentration of methyl pyropheophorbide 3 was 5000 nmoles/m1 of Triton X-100 solution. The Triton X-100 solution with methyl pyropheophorbide 3 was then added to the cultures to give a final concen- tration of 0.02% Triton X-100 and 500 nmoles methyl pyropheophorbide 3/ml in the culture. An equivalent amount of 2% Triton X-100 without methyl pyropheophor— bide was added to the control culture. Samples were taken periodically after addition of the compound. To measure chlorophyll and methyl pyropheophorbide 3 con— tent of the cells, the equations described by Vernon (1960) were used. Since the visible absorption spectra of pheophytin 3 and methyl pyropheophorbide 3 are identi- cal, it was assumed in this experiment that the pheophytin 3 concentration calculated with the equations was in fact methyl pyropheophorbide 3. Methyl pyropheophorbide 3 was prepared from leaves of Ailanthus altissima. Methyl chlorophyllides were prepared by the method described by Holt and Jacobs (1954) using methanol rather than ethanol. The method is based on transesterification of phytol with methanol catalyzed by chlorophyllase. Methyl chloro- phyllides 3 and 3_were converted to pheophorbide 3_and 3 21 and separated by their difference in HCl number. Por- phyrins of low HCl number were first washed out of an ether solution of the pigments with 10% HCl. This treat- ment converted the chlorophyllides to pheophorbides. The methyl pheophorbide 3, formed during the first HCl extraction, was then extracted with 17.5% HCl. Methyl pheophorbide 3 remained in solution. Methyl pheophor- bide 3 was suspended in a 50:50 benzene-petroleum ether solution and chromatographed on a sugar column with the same solution. Pyrolysis was carried out according to the method of Pennington 3E_31. (1964). A sample of methyl pheophorbide 3 (SO-100 mg) is dissolved in 10 ml of pyridine and heated in an evacuated ampule at 100°C for 24 hours. Purity of the compound was tested by thin layer chromatography. The compound was then trans- ferred to ether, dried, and suspended in 80:20 ether- acetone to give a concentration of 300 nmoles/0.05 m1. Concentration was measured by using molecular absorption data given by Pennington 33 31. (1964) for ether. They reported the molecular absorption coefficient for methyl Perpheophorbide 3 at 667 nm to be 52,000 M"1 cm‘l, CHAPTER IV RESULTS Greening Under Normal ConditIons Greening of the cells was achieved by transfer- ring previously bleached cells into basal medium con- taining 0.5% urea and exposing them to continuous light. Figure 1 shows the changes in chlorophyll content and chlorophyllase activity after urea was added and cells were placed in the light. The results are expressed on the basis of a liter of culture. It can be seen that a lag of approximately 4 hours occurs between the time chlorophyll begins to increase and the time chlorophyllase activity increases appreciably. However, there is a measurable amount of chlorophyllase activity in the totally bleached cells. It is possible that this level of activity may account for the syn- thesis of chlorophyll during the lag period. The lag period varied throughout the greening experiments from 0 to 12 hours. The experiments were stopped as soon as the culture appeared deep green in color regardless of 22 23 mmmaamamOHoHno Olllll. .Hamcmoungo Olllllo .moHDOm cmmouufic mm mow: wm.o mchHmu Icon Edflcmfi Hmmmn cw DMHHDDHUU an ©m>mwnom mmB mHHmo muHQB mo mcfl Icmonw .mmcfloownuouonm .0 mo maamo mcfismmsm CH musuaso mo Hmuwa mom mwmcmzo mufl>flpom mmmaamnmouoHno can HHMQQOHOHQUII.H musmflm 24 NMOLES PHEOPHYTIN HYDIHRIL x 162 f g 2 O '0 '0' m T l r I F 1 I 20 / l2 TIME HRS no.1 L 1 fl .- 1I9fl 'l'lAHdOHO'Il-IO 25 chlorOphyll content or chlorophyllase activity. It was expected that any relationships between chlorophyllase and chlorophyll synthesis would be apparent by the time the culture was green. Figure 2 shows the changes in chlorophyllase activity and chlorophyll content expressed per 109 cells. The lag between the time of beginning chlorophyll syn- thesis and increase in chlorOphyllase activity is no longer apparent. When results are expressed in this way, they appear similar to those described by Chiba 3E 31, (1967). Bleaching Under Normal Conditions Bleaching of the cells was achieved by transfer- ring green cells to basal medium containing 1% glucose and incubating them in the desk. Figure 3 shows changes in chlorophyll content and chlorophyllase activity after the addition of glucose and removal from the light on a per liter of culture basis. There is little loss of chlorophyllase activity during the time when most of the chlorophyll is degraded. Results in Figure 4 focus on the same variables but on the basis of 109 cells. The results again appear very similar to those reported by Chiba 3E 31. (1967), while those in Figure 3 are clearly different. There was a four-fold increase in cell number during the 26 mmmaamnmonoHno Olllllo .Hawsmouoano Olllllo .lensmfim ca mm mEmm was mums poms Icou .mwcwoomnuouonm .0 mo mHSDHDU mcwsmonm m Cw maaoo mmmcmno MDH>HDUM wmmaamsmouoHao can HHmSQOHOHQUII.N musmwm mcoflpflm m OH Mom NMOLES PHEOPHYTIN H YDIHR o 2 I 27 N -. 9N 'I'IAHJOHO'IHO l2 IS 20 24 TIME HRS 28 mmmHHmsmouoHno .lllll. .HHMBQOHOHSU Olllllo .mousom sonumo m mm mmoosam wa msflcfimucoo EDHWmE oucw mHHWU Mmmum mcwunmmmcmuu an Um>mflcom was mafinommam .mmcwoomnuououm .0 m0 mHHmo mcwcomman CH musuaso mo kuHH mom mmmcmno mufl>wuom mmmaahsmonoHno can Hamsmouoanoll.m musmflm 29 NHOLES PHEOPHYT IN HYDIHRI L '0 O o 8 r qr L 8 'I'IAl-IdOHO'II-IO 32 40 48 TIME HRS 24 IS 30 mufl>fluom mmmaamnmouoHso OIIIIIO .HHSEQOHOHBU OIIIIIO .h musmwm CH mm meow mnu mums com: Icou .mwcHOUQSDODOHm .0 mo musuaso mcwnomman m CH mHHmo momsmno >DH>Huom mmmHHNBQOHoHso can HamnmonoHQUIl.v musmwm muesnflw Hmm moa 31 NMOI.ECS> PHEOPHOY TIN HY DIHRI '9 N T I 48 4O I 32 I 24 TIME HRS 1 IS ; 2 g on 'I'Iu-Idoumiw 32 bleaching period. Therefore, expression of the results on a per cell basis makes it appear that there is a parallel loss of both chlorophyll and chlorophyllase activity during bleaching. There is approximately a three-fold excess of chlorOphyllase to account for the rate of chlorophyll degradation. Assuming the 13_X123 rate of hydrolysis is approximately equal to the rate measured 1n_y1££o. EXperiments were terminated when cells became pale yellow in color. At this point, extraction of intracellular material was made difficult by the high lipid content of the cells. Also, the read- ings of chlorophyll concentration were affected because lipids would precipitate on the sides of the cuvettes. It should be noted that chlorophyllase does not disappear totally even though cells may be bleached for an extended period of time. Inhibitor Experiments Figure 5 shows the effect of cycloheximide (5.3 x lO-SM) on the greening of cells, the antibiotic was in contact with the cells 15-16 hours before addition ' of urea and exposure to light. A control under the same conditions was also run. Results are expressed on a per liter basis. As may be observed, cycloheximide completely inhibits the formation of chlorophyll. Chlorophyllase 33 Figure 5.--Chlorophyll and chlorophyllase activity changes per liter of culture in greening cells of C. protothecoides with 5.3 x 10-5M cycloheximide addEd to the greening medium. Cycloheximide was added to bleached cells in basal medium 16 hours prior to addition of urea. Changes in a control culture car- ried out at the same time and under the same conditions are also shown. O—-———O O—————O chlorophyll in control culture chlorophyll in cycloheximide treated cells chlorophyllase activity of the control culture chlorophyllase activity of the cyclo- heximide treated cells 34 5.2.9. $833... 523:. x an 0 0 0 4 3 2 w a q _ _ .1 p F . _ _ _ P J ~62 1.1;..30104 :u 24 36 48 TIME HRS {z 35 activity does not change significantly during the course of the experiment. Figure 6 shows the same results on the basis of 109 cells. The situation does not appear markedly different than when expressed on a per liter of culture basis. The control culture shows a typical lag between chlorOphyll synthesis and initial rise in chlorophyllase activity. It should be noted that although practically no chlorophyll exists, there is a measurable level of chlorophyllase activity in the experimental culture. Figure 7 shows the effect of cycloheximide on the bleaching of cells. Treatment was analogous to that of greening cells, but 1% glucose was added to the culture at the end of the 16-hour pre-incubation period and the cells were maintained in darkness. The results are expressed on the basis of a liter of culture. Cycloheximide stops bleaching after 24 hours completely. Figure 8 shows the same results on the basis of 109 cells, it seems to indicate a close relationship between chlorophyll degradation and loss of enzyme activity as indicated by Chiba 33 31. (1967). The higher chlorophyll content in the treated cells, at time zero, may be due to partial inhibition of bleaching by cycloheximide even in the dark. Figure 9 shows the effect of Chloramphenicol 2 (2 x 10- M) on greening cells. The inhibitor was 36 Figure 6.--Chlorophyll and chlorophyllase activity changes per 109 cells in a greening culture of g. protothecoides with 5.3 x 10‘5M cycloheximide added to the greening medium. Conditions are the same as those described in Figure 5. Control culture results are also shown. O——-——O chlorophyll in the control culture O————-O chlorophyll in the cycloheximide treated cells V V chlorophyllase activity of control culture V V chlorophyllase activity of cyclohexi- mide treated cells 37 0 7 zzornm v1n01:<._._z r202...» 0 6 0 5 O O O O 4 3 2 I - q _ A . d _ _ 36 21 TIME HRS 1114 1 I2 .I2 - _ 8 O .04 - a: w4>zmo¢oszu 38 Figure 7.--Chlorophyll and chlorophyllase activity changes per liter of culture in bleaching cells of C. protothecoides with 5.3 x 10‘5M cycloheximide added to Ehebleaching medium. Cycloheximide was added to green cells in basal medium 16 hours prior to the addi- tion of glucose. Changes in a control culture carried out at the same time are also shown. O-—-——O O-——-—O V chlorophyll in the control culture chlorophyll in the cycloheximide treated cells chlorophyllase activity of control culture chlorophyllase activity of cycloheximide treated cells 39 zxormm 2.82.3.2 :42sz 83» TIME HRS o 0 3 2 m . . _ o a I‘- O in .\ I“ 12 I .\ r u p n 6 S 4 3 2 I 4‘02. 44>13010410 40 Figure 8.--Chlorophyll and chlorophyllase activity changes per 109 cells in a bleaching culture of C. protothecoides with 5.3 x 10'5M cycloheximide addEd to the bleaching medium. Conditions are the same as those described for Figure 7. Control culture results are also shown. O-————O O—————O chlorophyll in the control culture chlorophyll in the cycloheximide treated cells chlorophyllase activity in control culture chlorophyllase activity of cycloheximide treated cells 41 +90 0 8 zzornm 2182.3... 13:; w 0 7 0 ab 0 O 3 _ ~20 -IO q 4 A o 24 3‘ 48 TIME HRS I2 42 Figure 9.--Chlorophy11 and chlorophyllase activity changes per liter of culture in greening cells of C. protothecoides with 2 x lO‘ZM Chloramphenicol added to Ehe greening medium. Chloramphenicol was added to bleached cells in basal medium 16 hours before addition of urea. Changes in a control culture carried out at the same time are also shown. O—————O o—-———o chlorophyll in the control culture chlorophyll in the Chloramphenicol treated cells chlorOphyllase activity in control culture chlorophyllase activity of Chloramphe- nicol treated cells 43 2233 252.13.: 32.32. .20.» m s .o. - q 1 P b L b _ s 4 3 z .l .302 44>IAO¢OJIU 20 24 IS _a-—_ .- I2 TIME HRS 44 added 15-16 hours prior to addition of urea and exposure to the light. The treated cells show an increase in chlorophyllase activity while no synthesis of chloro- phyll occurs. Figure 10 shows the effect of Chloramphenicol on the bleaching of cells. Concentration of Chloramphe- nicol was the same as that used for greening cells. Addition of Chloramphenicol to the cells already in the dark, was 15 to 16 hours prior to adding glucose. Cells were kept in the dark during the experiments. Results are expressed on the basis of a liter of culture. Figure 11 shows the effect of Chloramphenicol on the bleaching of cells, this time expressed on the basis of 109 cells. No significant differences between the treated cells and the control are noticeable. Figure 12 shows, on the basis of a liter of culture, the effect of methyl pyropheophorbide 3 on the chlorophyll content and chlorophyllase activity during the greening of cells. Methyl pyropheophorbide 3 was added after 24 hours of incubation in the dark. The compound was dissolved in a 2% Triton X-100 solution. The final concentration of Triton X-100 in the medium was 0.02%. The final concentration of methyl pyropheo- phorbide 3 in the medium was 500 nmoles/ml. Control cells were also grown in a medium containing 0.02% Triton X-100. 45 Figure lO.--Chlorophyll and chlorophyllase activity changes per liter of culture in bleaching cells of C. protothecoides with 2 x 10’2M Chloramphenicol added to {he—bleaEhing medium. Chloramphenicol was added to green cells in basal medium 16 hours before addition of glucose. Changes in a control culture carried out at the same time are also shown. O-————O O———-—O V chlorophyll in the control culture chlorophyll in the Chloramphenicol treated cells chlorophyllase activity in control culture chlorophyllase activity in Chlorampheni- col treated cells 46 Z‘Ormw V1MO$I<120=I¢O¢OJIU d as 24 TIME HRS I2 51 The chlorophyll content of the treated cells behaves much like the control culture, but chlorophyllase activity of treated cells rises sharply 6 hours earlier than the control. Figure 13 shows the results expressed on the basis of 109 cells. Basically the same pattern is observed. The significance of this early rise in chlorophyllase activity is not clear at this time. V. Figure 14 shows the effect of methyl pyropheo- phorbide a on the chlorophyll content and chlorophyllase activity of bleaching cells on the basis of a liter of % culture. One observes that chlorophyll degradation occurs at about the same rate in both cultures. Chloro- phyllase activity, however, is noticeably different. While a consistent decline of enzyme activity cells occurs, activity increases in the treated cells. Figure 15 shows that on the basis of 109 cells the results are similar to those in Figure 14. Low initial activity of the enzyme in treated cells may be due to the effect of residual ether-acetone at the time of adding the compound to the culture. The compound was dissolved in ether—acetone (80:20) and then suspended in a 2% Triton X-100 solution. The ether-acetone was evacuated at low pressure. Large losses of the compound were unavoidable unless some of the ether-acetone remained in the Triton X-100 solution. 52 Figure l3.--Chlorophyll and chlorOphyllase activity changes per 109 cells in a greening culture of C. protothecoides with 500 nmoles of methyl pyropheophorbide a per milliliter of culture medium added. Conditions are the same as those described for Figure 12. Control culture results are also shown. O—-—-—O chlorophyll in the control culture O-————O chlorophyll in the methyl pyropheophor- bide 2 treated cells V V chlorophyllase activity in the control culture V V chlorophyllase activity in methyl pyropheophorbide 3 treated cells 53 zxormm 11m011