WWWWW " LIBRARY Michigan State THEs$ .IJQXVCCHCY _' This is to certify that the thesis entitled THE BIOSYNTHESIS OF A-AMINOLEVULINIC ACID AND TETRAPYRROLES AND THEIR REGULATION IN GREENING BARLEY: INVESTIGATIONS WITH METABOLIC INHIBITORS presented by PETER WILLIAM BERGUM has been accepted towards fulfillment of the requirements for Master of Science . Botany and Plant —_.degreem logy D" Major professor Date . 5 CI 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. THE BIOSYNTHESIS OF A-AMINOLEVULINIC ACID AND TETRAPYRROLES AND THEIR REGULATION IN GREENING BARLEY: INVESTIGATIONS WITH METABOLIC INHIBITORS By Peter William Bergum A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1979 fl,-_ ‘a-a v4.-‘._5fi__v_»4k,-__..r 44 .— ‘—‘O‘_——‘ ., ‘me—Ho. H~—m- ~ ._i__,-— "-ruw—< .......--» .... m 1..."-.- ABSTRACT THE BIOSYNTHESIS OF A-AMINOLEVULINIC ACID AND TETRAPYRROLES AND THEIR REGULATION IN GREENING BARLEY: INVESTIGATIONS WITH METABOLIC INHIBITORS By Peter William Bergum The effect of isonicotinic acid hydrazide on the biosyn- thesis of chlorophyll in greening barley was investigated. Isonicotinic acid hydrazide is an antagonist of pyridoxal phosphate and as such can be used to detect such pyridoxal phosphate-dependent enzymes as d-aminolevulinatic acid synthase. The hydrazide inhibited S-aminolevulinate formation but appeared to have an additional inhibitory site in the choro- phyll biosynthetic pathway. It caused an increased accumula- tion of protoporphyrin with a concomitmant decrease in pro- tochlorophyllide in aminolevulinate-treated seedlings. Labelling experiments with 14C-aminolevulinate not only indi- cated a site of action for the hydrazide between protoporphy- rin and protochlorophyllide but also suggested an increased protoporphyrin turnover. Pre-treatment with light or nicotin amide attenuated these effects of isonicotinic acid hydrazide. These observations are consistent with a dual inhibitory action of isonicotinic acid hydrazide on chlorophyll synthesis: it appears to act on 6-aminolevulinate synthesis as a pyridoxal Peter William Bergum phosphate antagonist and on tetrapyrrole synthesis via an effect on the pyridine nucleotide pool. Aminolevulinate synthase does not appear to play a major role in d-amino- levulinate synthesis in greening barley. ”. . . Chlorophyll . . . is the real Prometheus, stealing fire from the heavens." Timiryazev ii This thesis cannot be dedicated to just one person. I would therefore like to dedicate it with the sincerest gratitude and deepest heartfelt feelings to - My parents, Dr. Frank A. Loewus, Doug, Yael, Mona, and the memory of Ernest Erickson - who, each in their own very special and unique way, have given so much not only to the realization of this thesis but also to me through their support, encouragement, and understanding. ACKNOWLEDGMENTS It is very difficult, due to the limitation of space, to include all these people who have helped me in so many ways during my graduate studies here at Michigan State. For these peOple, I would like to express a special thanks. For my interest in plant science, I am deeply indebted to Dr. Frank A. Loewus, my undergraduate adviser. His enthusiasm for plant biochemistry, his support and encourage- ment were inspirational for me in pursuing a graduate career in plant physiology. I would also like to thank two of the best lab colleagues one could ever possibly have - Drs. Yael Avissar and Ronald Myers for providing a delightful atmosphere for scientific work and intellectual discussion. I am also grateful for their undying support when it was needed and their friendship. I thank Dr. R. S. Bandurski for the use of his spectro— photometers and Dr. C. P. Wolk for generously providing me "refuge" in his laboratory during the writing of my thesis. To the women of Dr. Wolk's lab, I owe a warm thanks for pro- viding such a pleasant atmosphere for writing my thesis - even if all the distractions didn't help me finish faster writing my thesis. iv To Dennis, Nien-tai, and Adiva, I thank them for their "sympatheic ears," continuous moral support and friendship. I'm eternally grateful to Yael for all her very useful suggestions for the revision of my thesis, for the many stimulating intellectual exchanges not only about science but about literature, art and life, and for our very special friendship. I would like to thank the members of my committee, Drs. Philip Filner and Norman Good for their critical review of my thesis work. I am grateful to my advisor, Dr. Kenneth Nadler, for providing me with the opportunity to grow as a scientist. I believe that we both have learned a great deal from one another. I will be forever grateful to my best friend, Doug, who was able to ”put up” with me during this entire time. His constant support, limitless understanding and caring have shown me that there can be no better friend. I am thankful to Barb for her excellent expertise in graphics, her listening ears and heart, and her warm friendly smile. And lastly I thank Mona, who bore with patience, under- standing and love our separation and my frequent "silence" over so many miles. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES. LIST OF ABBREVIATIONS. INTRODUCTION. The Biosynthesis of 6- Aminolevulinic Acid (ALA) Alternative Pathways for ALA Synthesis The Existence of Multiple Pathways in Barley For the Formation of ALA . The Biosynthetic Pathway Chlorophyll and Other Magnesium Tetrapyrroles. . . Regulation of Chlorophyll Biosynthesis Isonicotinic Acid Hydrazide (INH) The Use of Isonicotinic Acid Hydrazide in an Attempt to Determine the Presence of d-Amino- levulinic Acid Synthase in Greening Barley. MATERIALS AND METHODS. Growth of Seedlings. Extraction and Pigment Determinations. Identification, Separation, and Fluorimetric Quantitation of Tetrapyrroles. . . Extraction and Determination of ALA Radiochemical Procedures Measurement of Respiredl 4C02 . Incorporation of [4C] ALA into Tetrapyrroles Colorimetric Assay for Isonicotinic Acid Hydrazide Experimental Procedures for the Synthesis of [14 C]- ALA in Blue-Green Algae. . . . . . . RESULTS vi Page ix xi xiii (NH 11 12 19 20 23 23 24 25 28 28 29 29 30 32 A. Effect of Isonicotinic Acid Hydrazide on 5-Amino— levulinic Acid and Chlorophyll Formation . Variations in Greening Capacity The Effect of INH . . . Attempts to Reverse the INH~ inhibition of Chlorophyll Synthesis . The Effect of INH on ALA- stimulated Tetra— pyrrole Synthesis . 5. Nicotinamide - An Antagonist of the INH- inhibition of Tetrapyrrole Synthesis 4> LNNH B. Comparative Effects of Other Metabolic Inhibitors on 6— Aminolevulinic Acid and Tetrapyrrole Synthesis . . . . l. Analogs of INH . . 2. Additional Inhibitors. 3. Arsenite: Effect on ALA and. Chlorophyll Formation C. Photorespiratory Inhibitors: Their Effects on ALA and Chlorophyll Formation D. The Effect of INH on the Synthesis .of 6- -Amino- levulinic in Anabaena variabilis. . . . DISCUSSION. I. The Effects of INH on Tetrapyrrole Biosynthesis in Greening Barley Shoots II. The Reversal of the INH- inhibition of Tetra- pyrrole Biosynthesis by Light, ALA, and Nicotinamide . . . . A. Reversal by Light B. Reversal by ALA. C. Reversal by Nicotinamide. III. The Mechanisms of INH Action on the Biosynthesis Of Tetrapyrroles and ALA. A Hypothesis. A. Mechanism of INH Action on the Biosynthesis of Tetrapyrroles. . A Possible Mechanism of Action for INH: The Interaction of the INH-NAD Analog and the Pyridine Nucleotide Pool and Its Relation to Light . . . . . . . Page 32 32 32 45 50 69 74 74 78 8O 83 88 92 93 108 108 110 111 112 114 119 B. Mechanism of INH Action on the Biosynthesis of é—Aminolevulinic Acid . . . 6-Aminolevu1inic Acid Synthase (ALAS): Does It Exist In Greening Barley Shoots? . IV. Additional Modes of Isonicotinic Acid Hydra- zide Action. . . . . . . . . . A. INH as a Metal Chelator . B. INH as a Photorespiratory Inhibitor of the Glycolate Pathway V. Summary. LITERATURE CITED. APPENDIX viii Page 122 131 133 133 134 136 138 150 LIST OF TABLES Table Page 1. Effects of various amino and organic acids on the inhibition of chlorophyll synthesis by INH: an attempt to reverse the inhibition . 46 2. Effects of INH on the synthesis of tetrapyrroles and carotenoids in low light in the presence of ALA. . . . . . . . . . . 62 3. Effects of INH on the radiospecific activity (* SA) of accumulating PROTO and PCHLD in barley shoots fed ALA in the dark . . 64 4. Effect of INH on the *SA and synthesis of tetrapyrroles derived from 14C— ALA in the light . . . . . . . . 67 5. The reversal by nicotinamide of the inhibitory effects of INH on the *SA and synthesis of tetrapyrroles. . . . . 7O 6. Effect of pre- treatment and treatment with nicotinamide or pyridoxine on the inhibition by INH of ALA formation in low light . . . 72 7. Effect of analogs of INH on ALA and chlorophyll biosynthesis . . . . 75 8. Effect of various inhibitors on ALA-stimulated tetrapyrrole synthesis: possible modes of action similar to INH . . . . . . . . . 79 9. Effect of INH on arsenite- stimulated ALA formation . . . . . . 84 10. Effect of arsenite on ALA— stimulated tetrapyrrole formation in the dark . . 84 11. Effect of various inhibitors and intermediates of the photorespiratory glycolate pathway on the formation of ALA and chlorophyll . . . 86 ix 12. 13. 14. Effect of INH on 14C0 -evolution from barley seedlings fed labelled amino acids . . . 87 Effect of AsOi, a-HPMS, and glycine hydroxamate on 14C02-evolution . . . . . . . . . 89 Effect of INH on the biosynthesis of ALA from 4C-precursors in Anabaena variabilis . . . 90 LIST OF FIGURES Figure 1. 10. 11. 12. The proposed pathways of 6- aminolevulinic acid biosynthesis . . . . . . . . . The biosynthetic pathway of chlorophyll and heme. . Structure of isonicotinic acid hydrazide and two physiologically important analogs. Time course of 6- aminolevulinic acid and chloro— phyll formation during the greening period. Effect of varying seedling age . . Effect of isonicotinic acid hydrazide (INH) on chlorophyll formation . . . . The effect of various concentrations of INH on the inhibitions of chlorophyll and ALA synthe- sis in low and high light. The effect of increasing light intensity on the inhibition by INH of ALA synthesis. . Accumulation of INH in barley tissue under low and high light . . . . . Reversal by ALA of the INH- inhibition of chloro— phyll synthesis An absorption spectrum of an ether extract of barley seedlings treated with both INH and ALA . . . The effect of INH on the increase in ALA- induced protoporphyrin (PROTO)and protochlorophyllide (PCHLD) accumulation in the dark . The stimulation of ALA-dependent PROTO accumula- tion by low concentrations of INH in the dark . xi Page 10 17 34 36 39 42 44 49 S3 55 57 Figure Page 13. The stimulation of ALA-enhanced PROTO accumula- tion by low concentrations of INH in low light . . . . . . . . . . . . . . 60 14. Effect of isonicotinic acid (INA) on chlorophyll synthesis. . 77 15. Effect of arsenite on ALA and chlorophyll synthesis. . . . 82 16. Proposed sites of action of INH on the bio- synthetic pathway leading to chlorophyll . . 96 17. Potential sites for INH action on the bio- synthetic pathway between protoporhyrin and chlorophyll . . . . . 117 w 18. Potential sites for INH action on 6-aminolevu1- inic acid biosynthesis in greening barley . . 130 ALA ALAS CHLD(D) CHL DOVA d-HPMS INA INH LEV MgPROTO MgPROTO ME NAmD PALP PROTO PCHLD PXN *SA SAM LIST OF ABBREVIATIONS 6—aminolevulinic acid 6-aminolevulinic acid synthase (EC 2.3.1.37) chlorophyll(ide) chlorophyll y, 6-dioxovaleric acid a-hydroxy-2-pyridine-methane sulfonic acid isonicotinic acid isonicotinic acid hydrazide potassium levulinate magnesium protoporphyrin magnesium protoporphyrin monomethyl ester nicotinamide pyridoxal phosphate protoporphyrin protocholorophyllide pyridoxine radiospecific activity S-adenosyl methionine xiii T'T—“T INTRODUCTION The Biosynthesis of 6-Aminolevulinic Acid (ALA) 6-Aminolevulinic acid (ALA) is the first intermediate com- mitted to the biosynthetic pathway leading to such tetrapy— rroles as heme, chlorophyll, bilins, and vitamin B12 (37). Like most other tetrapyrroles found in various organisms, chlorophyll is synthesized by the condensation of eight mole- cules of ALA (75). In bacteria, including photosynthetic bacteria, and in animal tissue, ALA formation is catalyzed by the pyridoxal phosphate-dependent enzyme, ALA synthase (ALAS) [succinyl CoA: glycine C-succinyl transferase (decar- boxylating), EC 2.3.1.37] (37). In animals, ALAS is found within the mitochondrion, existing close to a source of its substrate succinyl CoA and its end—product, heme, This close proximity of ALAS and heme may play an important role in the feedback regulation of the pathway. In Rhodopseudomonas spheroides, ALAS is feedback inhibited by heme (20); in addi- tion, there is some evidence to suggest that heme may also repress ALAS formation (68). In cultured liver cells, ALAS is subject to induction (96a), repression (96b), and product inhibition by protoporphyrin and heme (18, 37). In contrast to the well-elucidated model for ALA synthesis in bacteria and animals, a clear understanding of the mech- anism of ALA formation in higher plants remains elusive. It has been long argued that chlorophyll is formed by a pathway identical to that of heme synthesis in bacteria and animals - the only difference being at the metal chelation step. The existence of ALAS in plants - in contrast to its proven exis— tence in animals - is a subject of much controversy (cf 5, 85). There have been scattered demonstrations, appearing in the literature, of a "presumed" ALAS in such tissue as callus culture of soybeans (119) and greening potatoes (89, 90), even though the validity of such reports has been called into question (for a review: Beale, 5). The evidence supporting the presence of ALAS in plants is usually based on one of two criteria: 1) a glycine- and succinyl GOA—dependent formation of ALA in extracts of plants, or Z) the incorporation of label from 14 C—glycine or -succinate into chlorophyll or ALA. Most investigators have not shown that the ALAS activity which they were reporting, was actually glycine— and succinyl CoA— dependent (cf 119). When the presence of ALAS was concluded from the incorporation of l4C—glycine or -succinate into chlorophyll or ALA, careful controls were not reported - such as to whether C—2 of glycine was preferentially incorporated into C—5 of ALA or whether glycine and succinate were incor- porated significantly better than any other intermediate common to general metabolism (see 5, cf 48, 118). In opposi- tion to this criticism, however, there are several reports which suggest the presence of ALAS in certain greening tissues. Ramaswamy and Nair (89, 90) demonstrated an ALAS activity requiring ATP, MgClz, pyridoxal phosphate, succinyl CoA thiokinase, glycine and succinate in greening potatoe skins. In addition, 14 C-labelled glycine and succinate were equally incorporated into ALA, whereas a-ketoglutarate and glutamate were not. Furthermore the incorporation of glycine was dependent on the presence of succinate and vice versa. Label- ling experiments with two green algae, Chlorella fusca (86) and Scenedesmus obliquus (62), suggest that the ALAS pathway for ALA formation, indeed, appears to be in operation in these organisms (see Figure 1A). Alternative Pathways for ALA Synthesis Since the existence of ALAS in higher plants has appeared equivocal, and many investigators have been unable to find ALAS in a vast number of greening plants, which under normal conditions make enormous amounts of chlorophyll, the possibi- lity of alternative pathways for ALA formation has been con- sidered. The work of Beale and Castelfranco (8, 9) showed that greening tissues of higher plants, such as cucumber cotyledons, 14 barley and bean leaves, will preferentially incorporate C— labelled OI-KG,'glutamate, and glutamine into ALA compared to l4C-glycine or 14 C-succinate. Furthermore, they were able to demonstrate that the carbon skeleton of these five carbon precursors was incorporated intact into ALA. When the labelling Figure l. The preposed pathways of 6-aminolevulinic acid biosynthesis. Pathway A - Pathway B - Pathway C - the classical or ALA synthase pathway involves the pyridoxal phosphate-dependent enzyme, ALA synthase, which condenses succinyl CoA and 14 glycine. In the conversion of l- C-glutamate to ALA via the ALAS pathway, the label is 14 evolved as CO 2. the DOVA transaminase pathway involves a transamination of y, 6 -dioxovaleric acid (DOVA) to ALA. Ketoglutarate as opposed to glutamate is the more immediate precursor of ALA in this pathway. In the conversion of 1-14C-glutamate to ALA via the DOVA transa- minase pathway, the label appears in C-5 of ALA. the glutamate-l-semialdehyde aminotransferase pathway involves the transamination of glu- tamate-l-semialdehyde to ALA. Glutamate as Opposed to a-ketoglutarate is the more imme- diate precursor of ALA. In the conversion of 1-14C-g1utamate to ALA via the glutamate-l- semi-aldehyde pathway, the label appears in C-5 of ALA. m >: z mha III OI III 82. ab< IZIU mka on _ .urrr +N «5 AI me A Na. I? I N: mmIma4<_zmm N _ uwzmn N w N W poxo_au_»‘a mpa N N mpa IN A INA mwfiuaowmoexo I Ni. NI mmoa NI szazmama-xaom£wz4o <00 4>z_uu:m mpm +_ A mapr .uumn NI A NIW m NIH“ N 22 ogjiflozczfa NIH“ ~93on 8.-.. NI mmm0_m min: DZ< ._._>I._O~_OIU 11 and initial stages of proline biosynthesis. In these cases, however, the glutamate-S-semialdehyde is formed instead of glutamate-l-semialdehyde. The evidence for the glutamate-l- semialdehyde aminotransferase pathway is as follows: 1) soluble protein preparations from plastids which support ALA synthe- sis from 14 C (U) - glutamate in the presence of ATP, NADPH, and Mg2+ were also capable of synthesizing ALA from glutamate- l-semialdehyde in the absence of added cofactors, 2) the transaminase inhibitors aminooxyacetate and cycloserine in— hibited ALA formation from glutamate—l-semialdehyde, 3) B- hydroxy—glutamate, an analog of glutamate and potent inhibitor of the conversion of 14 C-glutamate to ALA, had no effect on the conversion of glutamate-l-semialdehyde toALA, and 4) the column fractions which possessed the ability to synthesize ALA from labelled-glutamate corresponded to the fractions containing the aminotransferase activity. These results suggest that an aminotransferase catalyzing the formation of ALA from glutamate—l-semialdehyde is likely involved in the synthesis of ALA in barley. The Existence of Multiple Pathways in Barley For the Formation of ALA The finding that both the ALAS and C-5 pathway exist together in a greening organism, Scenedesmus obliquus, raises the possibility that both pathways may exist in higher plants (54, cf 85). In Scenedesmus, it appears that ALA is formed predominantly via the ALAS pathway, but both pathways "co-exist” — 12 simultaneously and lead to chlorophyll formation. On the other hand, Stobart and Hendry (104) have suggested that that the ALAS pathway is in operation only in the dark in barley, while the glutamate pathway operates in greening leaves. Since they obtained incorporation of labelled-glycine into ALA in the dark, it was suggested that this might be mitochondrial heme synthesis as there was little chloroplastic formation of tetrapyrroles in the dark (cf 5). The fact remains however that heme synthesis appears to occur via the C—5 pathway in barley leaves, and regardless of how poor of a radioactive precursor a compound was, heme and chlorophyll were labelled equally, suggesting that only one pathway, the C-5 pathway, gave rise to both metallo-tetrapyrroles (22). The presence of two pathways in higher plants, both of which synthesize ALA, remains controversial. The Biosynthetic Pathway Chlorophyll and Other Magnesium Tetrapyrroles The first step in the biosynthesis of chlorophyll (CHL) (Figure 2) is the rate—limiting formation of ALA — discussed in detail in the previous section. Following ALA synthesis, two molecules of ALA are condensed to form the monopyrrole porphobilogen (PBG) by the enzyme ALA dehydrase. PBG is the monomeric pyrrole which serves as precursor of the subsequent porphyrin compounds. Four molecules of PBG are combined by the coordinated action of two enzymes, PBG-deaminase and uro— porphyrinogen III oxidase to protoporphyrin IX (PROTO), the 13 last intermediate common to both heme and CHL biosynthesis. After the synthesis of PROTO — at the point of metal insertion - the pathway bifurcates into the branches of heme and chloro- phyll. If Fe(II) is inserted into PROTO by ferrochelatase, heme is formed. If Mg(II) is incorporated into PROTO by Mg chelatase, Mg protoporphyrin (MgPROTO) is formed. In its conversion to chlorophyll, the newly synthesized MgPROTO under- goes a series of structural modifications which include the closure of a fifth ring and the addition of a phytol side chain as well as a photoreduction of one of the pyrrole rings. The first modification of MgPROTO occurring is on the carboxyl group of the propriate residue of ring C. This residue is esterified with a methyl group derived from S-adenosyl methio- nine. The transfer is catalyzed by MgPROTO IX methyl trans- ferase, yielding MgPROTO monomethyl ester (MgPROTO MB). In the events prior to ring closure, the activated proprionate residue undergoes a biosynthetic B-oxidation; the sequence for which is as follows: the B-carbon is desaturated, and hydroxylated yielding the acrylic methyl ester. The acrylic derivative is then oxidized to a ketone at the B-carbon, activating the a-carbon. This a-carbon condenses with the y— methane bridge carbon of the Mg-tetrapyrrole, yielding the cyclopentanone ring (for review: 16, 23, 27). The resulting tetrapyrrole is called Mg-Z, 4- divinyl pheoporhyrin a5 ME and belongs to a group of Mg—tetrapyrroles which contain this fifth ring called phorbins. The reduction of the vinyl side chain on ring B of the phorbin macrocycle to an ethyl group 14 is required to form Mg 2-vinyl pheoporphyrin a5 ME, or pro- tochlorophyllide (PCHLD) - the naturally accumulating pigment of dark-grown angiosperms. PCHLD, in subsequent reactions, will be photoreduced and phytylated to give chlorophyll. Treatment of dark-grown leaves of angiosperms with exogenous ALA gives rise to the accumulation of a PCHLD species which is not transformable in light, that is, is not photoreduced to CHLD. This PCHLD absorbs maximally between 628nm and 636nm 628 (38, 106). The native, phototransformable species, PCHLD650 - with an absorption maximum at 650nm - is intercontertible with PCHLD628 species (52, 19, 38). The interconversion of PCHLD628 to PCHLD650 is dependent on the free sites available on the photoconverting enzyme, holochrome (38, 107). The attachment of PCHLD628 to the holochrome results in a PCHLD650 -holochrome complex (PCHLD—H). The actual binding of the PCHLD to the 628 holochrome is apparently responsible for the spectral shift from 628 to 650 (PCHLD650) and appears to require ATP and NADPH (49, 42, 19, 38). Normally, native PCHLD fully 650 saturates all available apoenzyme. Under conditions where PCHLD628 accumulates, its accretion is a consequence of the lack of available sites on the holochrome to which the PCHLD628 can bind (38). If brief flashes are given to convert the PCHLD650 to CHLD, PCHLD628 will be rapidly converted to PCHLD650 depending on how quickly the PCHLD628 saturates the holochrome binding sites (106, 108, 38). In the presence of 7,8 light, the A -double bond of ring D of the PCHLD 650 is reduced, generating CHLD678 (holochrome—bound). The actual ”r " - ’ 15 source of the reductant responsible for the photoreducing event is still a matter to be resolved. The holochrome has been proposed to act as a reductase, undergoing oxidation and reduction itself with NADPH as reductant (16). It has also been suggested that the light absorbed by the PCHLD chromophore is responsible for the photoreduction (see 91 for discussion). We can summarize the overall process as follows to correspond to the in 1119 spectral shifts [as proposed by Brodersen (19) and modified by Horton (49)]: 1) PCHLD + holochrome + NADPH 628 + ATP + PCHLD650 [enzyme-substrate-NADPH—ATP] complex. The ATP is not only necessary for photoconversion of PCHLD628 but also appears to stabilize the PCHLDéso-holochrome (49); 2) 650 complex]; 3) CHLD PCHLD + light energy + CHLD678 [enzyme-product-NADP + ADP + dark + CHLD and CHLD + dark + 678 684’ 684 CHL(D)672 [product + holochrome + NADP + ADP]. The last spectral shift is presumably the phytylation of CHLD to CHL, ] the last step in CHL synthesis (16). Contrary to what used to be believed about the phytylation I process, chlorophyllase does not appear to esterify free phytol to the proprionate residue of ring D of CHLD (cf reviews 16, 91). Results from kinetic and inhibitor experiments sug— gest that the phytylation of CHLD is a multistep pathway involving the reduction of a terpenoid alcohol adduct to a phytol side chain (93). The esterifying enzyme isolated from maize shoots were shown to affix geranylgeranyl pyrophosphate to CHLD giving CHL a- geranylgeraniol. The CHL-geranylgeraniol Figure 3. 16 Structure of isonicotinic acid hydrazide and two physiologically important analogs. INH is a structural analog of both pyridoxal phosphate and the nicotinamide moiety of the pyridine nucleotide, NAD+. 17 22_%o._._ axon}. +9.2 3 £232 £5292 2: _._ .__ ”:0! +/ @ :o. \ ouzommo- ~=z.o\ \ 0/10 - m :. goings»: Eva. 3:203:3— 18 ester if formed first; subsequent stepwise hydrogenation of this intermediate gives rise to CHL-dihydrogeranylgeraniol, CHL-tetrahydrogeranylgeraniol, and CHL phytyl ester (93, 94). Regulation of Chlorophyll Biosynthesis The pathway of chlorophyll biosynthesis appears to be regulated at three points (see Figure 2): 1) at the level of ALA formation, where control over the flux of the ALA into the pathway is exerted; 2) at the point of metal chelation of PROTO IX, where the flow into the branch pathways of heme and Mg-tetrapyrroles is regulated; and 3) at the site of photo- reduction of PCHLD to CHLD, where light regulates the conver- sion in angiosperms. (In gymnosperms and most algae, this conversion has no light requirements.) All of the enzymes of the CHL pathway are contained in the etioplast, although studies with inhibitor of protein synthesis show that they are synthesized by cytoplasmic ribosomes. With the exception of the ALA synthesizing enzyme(s) - which is light inducible - all of the biosynthetic enzymes needed for CHL formation are present in non—limiting quantities in the etiochloroplast (97). The availability of ALA is generally accepted as being the rate-controlling factor in CHL biosynthesis (37, 10). In addition, the enzyme responsible for ALA formation is believed to be under feedback regulation (110). For example, if a block occurs in the pathway, whether naturally as in the case of etiolated plants with the physiological accumulation of PCHLD, or genetically, as in mutants defective in CHL 19 biosynthesis, little of the tetrapyrrole intermediates prior to the blockage will accumulate (36). When ALA synthesis is examined under these "obstructing" conditions, it is found to be reduced and the accumulation of intermediates before the block occurs only when the plant is fed ALA (110). From these observations as well as from extensive analysis of Mendelian mutants blocked in porphyrin synthesis, it has been suggested that PROTO and PCHLD have a feedback regulatory function on ALA synthesis (111, 112, 76, 36). It has also been proposed that another feedback loop exists in the Mg-tetrapyrrole path— way from PCHLD to the step converting PROTO to MgPROTO in Chlamydomonas (112). Etiolated bean leaves treated with iron chelators, such as a,a'-dipyridy1, produce large amounts of MgPROTO ME and smaller amounts of MgPROTO, PROTO, and PCHLD as well as increased amounts of ALA (25). These results appear to indicate that the iron chelators, by making Fe(II) unavail- able, antagonize the feedback inhibition of ALA synthesis by some iron~containing compound probably heme. A regulatory role for protoheme has also been implied in the case of greening barley, since protoheme appears to turn over rapidly in the absence of net synthesis during rapid CHL accumulation (22). Isonicotinic Acid Hydrazide (INH) Isonicotinic acid hydrazide (INH) is an antagonist of pyridoxal phosphate-dependent enzymes such as transaminases 20 and decarboxylases (114). It is widely used as a chemothera- peutic agent in the treatment of tuberculosis in man. It is known, for example, that INH inhibits the mitochon- drial conversion of glycine to serine by glycine decarboxylase hithephotorespiratory glycolate pathway of plants (59, 87). INH also inhibits heme synthesis in both rat liver mitochon— dria (65) and rabbit reticulocytes (17); this effect of INH is due to its inhibition of pyridoxal phosphate-dependent ALA synthase (14). The inhibition of pyridoxal phosphate-dependent enzymes by INH is a consequence of hydrazone formation between the hydrazide of INH and the aldehyde of pyridoxal phosphate (14, 114) (Figure 3). INH is known to be oxidized to pyridine-4-carboxyaldehyde by the horseradish peroxidase/Mn2+/O2 system (124). In addi- tion, INH appears to be metabolized to isonicotinic acid in wild type and INH-resistant cell lines of tobaCCO callus (13). The Use of Isonicotinic Acid Hydrazide in an Attempt to Deter- mine the Presence of 6-Aminolevulinic Acid Synthase in Greening Barley In consideration of the question: whether or not the ALAS pathway exists in higher plants, we attempted to resolve the issue by using the metabolic inhibitor, INH. As an antagonist of PALP-dependent enzymes, INH should inhibit ALA formation, if ALAS plays an active role in the formation of ALA in greening barley (cf 14). We have, in fact, demonstrated that INH inhibits ALA formation; our experiments indicate however 21 that the inhibition does not appear to be due to the presence of ALAS. It is more likely due to the presence of a transa- minase activity involved in ALA synthesis. When it was dis- covered that INH acted on CHL synthesis beyond the point of ALA formation, it became important to redirect our study in clarifying the precise site and nature of this effect. Isoni- cotinic acid hydrazide was shown to inhibit the formation of PCHLD, at a site between PROTO and PCHLD. In light of the significant regulatory function that these two tetrapyrroles play in CHL biosynthesis - as feedback inhibitors of ALA syn- thesis - it was imperative to elucidate this particular effect by INH. If INH acted indeed as pyridoxal phosphate antagonist, then how could we explain the effect on tetrapyrrole synthesis, where no transamination reactions are known to be required? We attempted to compare the effects of INH to other inhibitors, whose modes of action were better understood. Comparison of INH action with the actions of known chemical blockers of the porphyrin pathway, showed that only ethionine operated like INH. Both apparently block tetrapyrrole synthesis between PROTO and PCHLD. We then directed our attention towards clarifying the mech- anism of this action of INH on tetrapyrrole synthesis. From our experimental results and reports in the literature, it seemed likely that INH may exert its inhibitory effect on tetrapyrrole synthesis by affecting the pyridine nucleotide pool, and consequently the redox balance of the plant cell. Our observations of the antagonistic interplay of light and 22 INH inhibition support such a hypothesis. This lead us to re-investigate the nature of the INH effect on ALA synthesis; it was discovered that the effect on ALA formation was not primarily via pyridine nucleotides. Attempts were then made to examine the effect of INH on the actual synthesis of ALA from labelled glutamate. Although glutamate has been shown to be incorporated into ALA in barley (6), preliminary attempts proved unsuccessful. We then turned our attention to an organism similar in metabolism to a higher plant chloroplast, that is, the blue-green alga Anabaena variabilis, which syn- thesizes ALA exclusively via the C-5 pathway. In Anabaena, INH appeared to inhibit the transamination of a—ketoglutarate to glutamate, thereby inhibiting the synthesis of ALA from q-KG but not from glutamate. We concluded then that ALA syn- thesis in Anabaena involved glutamate as the immediate pre- cursor of ALA, supporting the proposed glutamate-l-semialde- hyde pathway. These results coupled with additional inhibitor studies of ALA synthesis in barley suggest that INH inhibits ALA synthesis in a manner consistent with an antagonism of pyridoxal phosphate. The mode of action of INH offers us some insight into the biosynthetic requirements and regulation of the chlorophyll pathway — in spite of its apparent lack of specificity. MATERIALS AND METHODS Growth of Seedlings Barley seeds, Hordeum vulgare var. Larker, purchased from Donald Keinath, Caro, Michigan, were surfaced—disinfected for 1 minute in 1% NaOCl, and rinsed thoroughly in running tap water. The seeds were then soaked in a MgZ+-supplemented Hutner's nutrient medium (Appendix 1) for 3 hours, and were sown in a flat (56cm X 26cm X 6.5cm) containing vermiculite moistened with 2.5 liters of Hutner's medium. The seeds ger— minated within 3 days in a dark, humidistated (80%) room at 28°C. Six- to seven-day old etiolated seedlings were used, unless noted otherwise. Manipulations of Plant Tissue The seedling tops were harvested under a dim, green safe- light by cutting off the shoots just above the level of the bed. Seedlings (~8 g) were trimmed to a uniform length of 8 cm by excision of the bottom section of the shoots, and placed into 15 ml beakers containing 6 m1 of solution. The transpiration stream served as the means of uptake. For experiments conducted in the dark, a small fan was placed 30 cm in front of the seedlings. For the greening experiments conducted in light, the beakers — containing shoots and 23 ——'———'ii 24 and solutions — were placed in an illumination chamber (95cm X 65cm X 62cm). In this case, air was drawn across the shoots by a small fan positioned in the back wall of the chamber. Incandescent lights provided the illumination necessary for greening. The light intensities were determined by a Kettering Radiant Power Meter (LDC). For low light experiments, 2 bulbs (Sylvania) @ 7% watts were used, generating a light intensity of 3.0 x 103 ergs/cmZ/sec. For high light experiments, 4 soft white bulbs (General Electric) @ 100 watts were employed, 4 ergs/cmZ/sec of light. Constant ambient producing 9.0 x 10 temperature was maintained in the chamber. The shoots were preincubated for 1 hour - unless indicated otherwise - to ensure adequate uptake of experimental solutions into the leaves. Extraction and Pigment Determinations At the end of the treatment period, the seedlings were removed from their solution, blotted to remove any excess moisture, and 2.75-3.50 g portions were weighed out. The tissue was extracted into 50 ml of ice-cold (initially -20°C) alkaline acetone (9 volumes of acetone to 1 volume of .l M NaZCOB) in a Waring Blender for 1 minute at the highest speed setting; the acetone extract was suction filtered through Whatman No. 1 paper, and the trapped residual plant material was re-extracted with an additional 50 ml of alkaline acetone. The filtrates were combined and an aliquot of the filtrate was transferred to a test tube and clarified by centrifugation at a speed setting of 6 for 5 minutes in a table top centifuge. 25 The absorbances of the clear filtrate were determined with a Gilford 240 Spectrophotometer at the following wavelengths (in nm): 480, 539, 628, 645, and 663. The pigment concentra- tions of PCHLD and CHL were determined according to the equations given by Klein and Schiff (64) in mg/l: CHL = 12.2 A - 0.07 A628, and PCHLD = 28.7 A - 5.21 A 663 628 663; carotenoids according to (A480 + .114 A663 - 0.638 A645)/ for 250,000 and PROTO according to the derived equation 46.1 A539 9.2 A628 (Gough, 36). In addition, the absorption maxima of the pigments were confirmed using a Cary 15 recording spec- trophotometer. Pigment synthesis was expressed as net synthesis corrected for the pigment content of shoots grown in the dark. Identification, Separation, and Fluorimetric Quantitation of Tetrapyrroles The porphyrins were identified according to the methods of Duggan and Gassman (25) as follows: the crude acetone filtrate was extracted into anhydrous diethyl ether, the volume of which was reduced by bubbling gaseous N2 through the ether; the porphrins contained in the ether extract were then separ- ated by chromatography in the dark on thin-layer plates of silica gel (EM Laboratories, Inc.) in benezene-ethyl acetate— ethanol (lOO:25:25, v/v) (25). The individual bands of pig— ments were located by their red fluorescence under UV light and their Rf values recorded. Before the chromatogram dried out, the pigmented bands were scraped from the plates and 26 eluted with acetone-methanol (4:1, v/v). The particles of silica gel were separated from the solvent by centrifugation, and an absorption spectrum was taken of each pigmented super- natant with a Cary 15 recording spectrophotometer. Pigments were identified by a comparison of their absorption maxima and their mobilities (Rf) in this solvent system to those previously reported in the literature (25). A quantitated separation of the porphyrins was accomplished by thin-layer chromatography on silica gel plates in lutidine: H20 (100230. v/v) and NH3 vapor (from NH4OH) [a modification of the method as described by Gough (36)] and the separated porphyrins were measured by fluorimetry. This solvent system permitted an easy elution of PROTO from the silica gel - much better than from the benzene-ethyl acetate-ethanol system (data not shown). A 200 pl aliquot of the acetone filtrate was applied in a narrow band to a thin-layer plate; the tetra— pyrroles were then chromatographed. The separated pigment bands were scraped from the plates into an eluting solvent. Acetone: methanol (4:1) served as the eluent for metallo- porphyrins and methanol: 2 N NH4OH (4:1, v/v) for protoporphy- rin. Small test tubes containing the pigmented silica gel and eluent were placed in a covered, ultra—sonicator both for 30 minutes to facilitate the elution process. After centrifuga- tion, the concentration of tetrapyrrole in the supernatants was determined fluorimetrically with a Turner filter fluori- meter, model No. 111. For the quantitation of PROTO, Wratten filter No. 405 was used for excitation and No. 23A for emissions; 27 for the quantitation of PCHLD, filter No. 47B was used for excitation and No. 23A for emissions. The concentrations were calculated from standard curves prepared for PROTO and PCHLD from pure samples. The pure sample of PCHLD was obtained chromatographically from an extract of ALA-fed, dark—grown seedlings; the PCHLD was estimated spectrophotometrically. The disodium salt of protoporphyrin IX (Sigma Chemical) was used as the standard. Extraction and Determination of ALA ALA was estimated by the method of Mauzerall and Granick (79) as modified by Urata (109). Seedlings were incubated in 30 mM potassium levulinate, a competitive inhibitor of ALA dehydrase, permitting the accumulation of ALA as described by Beale (7). Shoot tissue (2.75 to 3.5 g) was extracted as before into 100 ml of cold, 5% trichloroacetic acid (v/v) followed by suction filtration. The crude filtrate was allowed to stand overnight at 4°C; the extract was clarified by centri- fugation at 10,000 x g for 15 minutes. The resulting superna- tant was then decanted and a 1 ml aliquot of the TCA superna- tant was adjusted to pH 4.6 with 1 m1 of 2 M Na-acetate buffer and combined with .1 ml of 2,4—pentanedione in a test tube. The test tube, containing the mixture, was stoppered with a glass marble and immersed in a boiling water bath for 15 minutes to form the ALA—pyrrole. One ml of modified Ehrlich's reagent was then added to 1 ml of the cooled solution, con— taining the ALA-pyrrole, and the absorbance at 555 nm was 28 determined 15 minutes later. The concentration of ALA-pyrrole was calculated using E$§m = 15,600. The identity of the pyrrole was confirmed from its absorption spectrum and the ratio of the absorbances at 525 nm and 555 nm (ASZS/A555 = 0.69). Radiochemical Procedures Measurement of Respired 14CO2 The effect of various inhibitors on respired 14 CO2 was measured according to the method of Beale (6). Two grams of seedlings were exposed to 4 hours of illumination in beakers containing water. The seedlings were then removed, blotted, and 3 cm segments excised from the greenest, middle sections of the shoots. The cut sections were placed tops up, main— taining the original orientation of the seedlings, into 2 ml solutions containing a labelled amino acid and inhibitor, where indicated. The employed radiochemicals, purchased from New England Nuclear: [2-14C] glycine (14.1 mCi/mmol); [1—14C] glycine (11.58 mCi/mmol); [3,4-14C] glutamate (14.2 mCi/mmol); and [1—14C] glutamate (48.75 mCi/mmol) were individually added to the incubation solution giving a label concentration of 0.5 uCi/ml. The beakers containing the seedlings and label were placed in glass jars (14.5cm x 7cm (diameter)) fitted with lids from which 2.1 cm glass fiber filter discs wetted with 0.1 ml of l N NaOH were suspended. At specified inter- vals, the filter discs were replaced, and the radioactivity 14 of the C-carbonate, formed from the 14CO2 trapped on the 29 discs, was measured by liquid scintillation with a Packard Tri—carb scintillation spectrometer Model 3003; counting efficiency for 14 C was 82.0%. The filter discs were placed in vials each containing 10 ml of Aqueous Counting Scintillant (Amersham) and allowed to remain in the scintillation cocktail at least 1 hour prior to counting. Incorporation of [14C]-ALA into Tetrapyrroles Four grams of etiolated barley shoots were placed in beakers containing 5 m1 of 10 mM ALA and 1.51 uCi [4-14C] ALA (51.5 mCi/mmol, New England Nuclear), and allowed to green for 8 hours under low light. The pigments were extracted, and two 200 pl aliquots of each extract were chromatographied in the lutidine: H20 solvent system. One chromatogram served in the pigment quantitation, as described before. The other was used in the determination of the specific activities of [14C]- PROTO, -PCHLD, -CHL. The individual pigmented bands were located under UV light, and the silica gel, containing the pigmented bands, was scraped from the chromatogram into Vials to which was added liquid scintil- lant. The radioactivity was measured by liquid scintillation as before. Colorimetric Assay for Isonicotinic Acid Hydrazide Since INH has a strong affinity for the Cu(II) ion (3), it will react with the Lowry reagents used for protein determination 30 (74) to give a blue color. The INH contained in 5% TAC ex- tracts, was estimated colorimetrically by the following pro- cedure: .2 ml of TCA extract was added to 1 m1 of Lowry reagent C [50 volumes solution A (2% NaZCO3 in 0.1 N NaOH) + 1 volume solution B (0.5% CuSO4, 1% potassium citrate in HZO)] for 10 minutes. Then 2 ml of Folin-Ciocolteau reagent (diluted 3x) was added. After 30 minutes, the absorbance at 500 nm was read. The concentrations of INH in the experimental samples were determined from a standard curve. Experimental Procedures for the Synthesis of [14C]-ALA in Blue-Green Algae The blue-green alga, Anabaena variabilis Kfitz (A.T.C.C. 29413) was grown autotrophically in an 8-fold dilution of the nitrogen-free medium of Allen and Arnon (2) at 30°C in a Micro- ferm Fermentor (New Brunswick Scientific) in the laboratory of Dr. C. P. Wolk (Plant Research Lab, Michigan State Univer- sity). The growing cultures were continuously illuminated by cool-white fluorescent lamps (General Electric) and aerated until a density of 0.5-1.5 ug of CHL/ml was obtained. Fila— ments of blue-green algae were separated from the medium by suction filtration through Whatman No. 4 filter paper. The cells were then thoroughly washed with distilled water and resuspended in 40 mM HEPES buffer (pH 8.0) to give a final concentration of 200-300 ug CHL/m1. Warburg flasks were used as the reaction vessels in these experiments. A folded piece of paper wetted with .1 m1 of l N NaOH was placed in the center 31 well to trap the respired 14C02. The following solutions - where indicated - were placed in the outer well in m1: 1 M levulinate, 0.1; 200 mM INH, 0.4; labelled precursor, 0.1; 1 mM cold precursor, 0.03; .2 M ATP, 0.1; cell suspension, 1; and water to give a total volume of 2 ml. The radiolabelled precursors used were: [14C(U)] glutamate (260 mCi/mol), [1-14C] glycine (11.58 mCi/mmol, [2-14C] glycine (14.1 mCi/mmol), [1,4-14C] succinate (51.3 mCi/mmol), and [14C(U)]d-ketoglutarate (287 mCi/mmol), purchased from New England Nuclear. The stock concentration of all radioactive precursors was 0.1 mCi/ml. The reaction vessels were stoppered and placed under illumina- tion for 3 hours with continuous agitation provided by a shaker. The reaction was stopped upon the addition of .4 ml 3 N perchloric acid. The reaction mix was then transferred to a test tube, and 15 ml 1 N KOH was added to raise the pH to ~2. The filter papers, containing the 14 C-carbonate, were also removed, placed into vials to which liquid scintillant was added, and the radioactivity counted. The acid-killed cells were permitted to stand overnight at 4°C. The cell mix was then centrifuged at 10,000 x g for 15 minutes to pellet the cell debris; the supernatant was then decanted. The ALA contained in the supernatant was then chromatographed on Dowex 50, and purified as the ALA—pyrrole according to the procedures of Beale (6). The radioactivity contained in the ALA-pyrrole was measured by liquid scintillation, as described previously. The amount of ALA was measured by chemical means as described earlier. RESULTS A. Effect of Isonicotinic Acid Hydrazide on 6-Aminolevulinic Acid and Chlorophyll Formation 1. Variations in Greening Capacity Barley seedling of different ages showed substantial variation in their capacity to synthesize 6—aminolevulinate (ALA) and chlorophyll (CHL). Preliminary examination of INH effects on chlorophyll synthesis confirmed the need for uni- formity in the age of the plant tissue as well as the ratio of the tissue mass to the volume of extracting solution (3 g of tissue per 100 m1 of extracting solution proved to be ideal). The accumulation of ALA and chlorophyll was assayed during a 20 hr illumination period (Figure 4). A significant differ— ence between 6%- and 7%-day old leaves could be seen in total levels of both ALA and CHL produced (cf Hendry a Stobart, 47). After 8 hr, the older tissue contained only 55% of the ALA and 63% of the CHL that the tissue a day younger did. Six-day old shoots were found to possess the highest synthetic capacity (both ALA and CHL) of all ages tested, and consequently were the seedlings of choice. 2. The Effect of INH Isonicotinic acid hydrazide effectively inhibited CHL accumulation by 85% during a 19 hr greening period (Figure 5). 32 Figure 4. 33 Time course of 6-aminolevulinic acid and chlorophyll formation during the greening period. Effect of varying seedling age. The ALA and chlorophyll content of 6%- and 7%-day old seedling were measured after various lengths of illumination. Seedlings used for ALA deter- minations were incubated in 30 mM levulinate, those used for chlorOphyll determinations were incubated in water. The light intensity was 5600 ergs/cmZ/sec. I 0—0 H ( NET CHLOROPHYLL CONTENT 34 6% contents in A nmol/g-fresh wt. 1 1 L l I 4 8 12 16 20 Hours of Illumination H O-AMINOLEVULINATE CONTENT Figure 5. 35 Effect of isonicotinic acid hydrazide (INH) on chlorophyll formation. Seven-day old barley shoots were incubated in either water or a 0.1 M solution of INH for 20 hours in a light intensity of 5600 ergs/cmz/sec. The chlorophyll content of the tissue was assayed after various times of illumination. 36 _ _ _ m m m m m w 8 6 4 2 16 2O 12 HOURS OF IL LUMINATION 37 The effect of INH was rather rapid: more than one half of the total inhibition was attained within the first 4 hr of greening. The potency of INH was clearly demonstrated through its inhibition of ALA and CHL synthesis in low light (Figure 6). Increasing the concentration of INH causes a dramatic rise in the inhibition of both ALA and CHL synthesis. At a con— centration of 20 mM, INH maximally inhibited CHL formation by 78% in low light, whereas ALA was inhibited only 52%. Since CHL synthesis was significantly more sensitive to the effects of INH than ALA synthesis, it suggested - as one possibility - a second site of inhibition for INH. The apparent Ki's for these inhibitions differed, being ~ 3 mM for CHL synthesis and ~ 10 mM for ALA synthesis. INH appeared, therefore, to be a more effective inhibitorIDfCHL than of ALA synthesis. The fact that both CHL and ALA synthesis were significantly inhibited, however, indicates that INH may act in part on ALA synthesis. The inhibitiory effects of INH appear to be dependent on light intensity. The inhibitions by INH (20 mM) of both ALA and CHL synthesis were reduced by 20—25% by increasing the light intensity from 3,000 to 90,000 ergs/cmZ/sec. From the curve (Figure 6), it was evident that the inhibitions never reach a plateau at any concentration of INH in high light - quite contrary to the effect observed in low light where the inhibition curves plateau sharply. This light effect was examined more closely: the efficacy of 10 mM INH was monitored at several light intensities from Figure 6. 38 The effect of various concentrations of INH on the inhibitions of chlorophyll and ALA synthesis in low and high light. Barley shoots were incubated in various concentra- tions of INH for an 8 hr illumination period under either low (L) light (3000 ergs/cmz/sec) or high (H) light (90,000 ergs/cmZ/sec). Seedlings destined for the chlorophyll assay were incubated in water and/or INH; seedlings used for the ALA assay were incubated in 30 mM LEV and/or INH. Inhibitions of synthesis represent the percent of CHL or ALA synthesis which was inhibited by INH as compared to the control. The controls for ALA, L and H in nmol/g-fresh wt. : 468 i 15, 781 i 37 and CHL, L and H in nmol/g-fresh wt.: 44.6 i 8, 101.1 20. + 39 (n—e) sgseqi UK; NM va 3!"!I""°l°u!'~"V-9 ASE: co=ozcmuc0u :2. oo— 00 ca on 00 on 9. on ON 9m 2301... no: a: Na 23: may. mzo=<_>un n¢: 52 2. 3.6.83 .3. u< 2.3a 50.50.39... EEoNufzI <._<-< >m ZO_._._m=._Z_ .. 12. m0 ._m~_ louuoa pewsu-HN' {o 0903 50 reversal of the inhibition (data not shown). The apparent concentration of ALA reversing one half of the total inhi- bition was ~5 mM, suggesting that INH does indeed inhibit the synthesis of ALA. A noteworthy observation was made concerning the appear- ance of shoots supplied with 10 mM ALA and 20 mM INH in low light; they showed a symptomatic wilt caused by a loss of turgidity that shoots fed ALA in high light also exhibited. The seedlings exposed more directly to the light - positioned closest to the sides of the beaker - appeared blanched com— pared to the greener, shielded tissue - closer to the inside. This was not observed in tissue treated with either water or ALA alone. This blanching effect is often characteristic of photosensitization (cf. 11, 113) and might be attributed to the accumulation of pigmented precursors of CHL. The INH- treated tissue also bore a faint brownish color, suggesting the accumulation of some brown pigment, presumably derived from ALA and a site of INH action beyond ALA synthesis. 4. The Effect of INH on ALA-stimulated Tetrapyrrole Synthe- To pinpoint additional, possible sites of action for this inhibitor, we examined the effects of INH on the syn- thesis of other intermediates of the biosynthetic pathway leading to CHL. We conducted these experiments in the dark - where little ALA and no CHL are formed - in the presence of added ALA. This experimental design enabled us to investi- gate that portion of the pathway between the formation of 51 ALA and PCHLD, the Mg-tetrapyrrole which accumulates in darkness. Spectrophotometric and chromatographic analyses of pigmented extracts from tissue incubated in ALA in the dark revealed the presence of only two tetrapyrrolic com- pounds: PROTO and PCHLD - even with added INH (Figure 10). The effect of 20 mM INH on PCHLD and PROTO formation was examined over the course of a 24 hr dark incubation period (Figure 11). The shoots were incubated in 10 mM ALA solu- tions to facilitate tetrapyrrole synthesis, which is quite low without light activation. During the first 9 hr, there appeared to be no significant differences in either PCHLD or PROTO synthesis between INH—treated and untreated seedlings [cf the light experiment, where CHL synthesis was inhibited 78% (Figure 5) and 40% in the presence of 30 mM ALA within 8 hrs (Table 1)]. Between 9 and 24 hr, INH inhibited PCHLD synthesis about 20% and seemed to slightly stimulate PROTO synthesis. These results implied a possible site of action between PROTO and PCHLD. In order to investigate this possi— bility further, various concentrations of INH were tested for their effects on ALA-stimulated tetrapyrrole synthesis in the dark (Figure 12). A11 concentrations of INH less than 20 mM stimulated PROTO synthesis between 17 and 31%, whereas PCHLD was inhibited between 0 and 20% over these same concentrations. At the higher INH concentrations (50 and 100 mM), synthesis of both tetrapyrroles from exogenous ALA was inhibited but not to the same extent; PROTO accumulation Figure 10. 52 An absorption spectrum of an ether extract of barley seedlings treated with both INH and ALA. Seedlings were fed 20 mM ALA in the presence of 20 mM INH for 21 hr in the dark. At the end of this time, seedlings were extracted into alkaline- acetone and filtered. The acetone filtrate was then extracted with diethyl ether to transfer the pigments into the ether phase. An absorption spectrum was recorded. The absorbance between 350-450 nm is given on the 0-1 absorbance scale; the absorbance between 450-700 nm is expressed on an 0.1 absorbance scale. Key to absorbance maxima (in nm): PhotochlorOphyllide: 432, 534, 571, 624 Protoporphyrin: 403, 503, 534, 575 Chlorophyll a: 663 Carotenoids: 469 53 E: E Ih02w4m><>> 005 can com 000 com 9.: 00¢ d SONVBHOSSV Figure 11. 54 The effect of INH on the increase in ALA- induced protoporphyrin (PROTO) and protoch- lorOphyllide (PCHLD) accumulation in the dark. Seedlings were incubated in 10 mM ALA i 20 mM INH for 24 hr in the dark. Uptake of the solutions was facilitated by air blown across the shoots from a small fan. At various times, seedlings were removed and the PROTO and PCHLD contained therein was extracted and quantified fluorimetrically. (See materials and methods for further details.) 55 ...ovos. 5 $85 08: 5:03.30:— VN N m— m— N— o o m 43 3.2.0:... 5 3.0.25? \« .2. :83 H .120 2:6. * \ .2. .0 02.0.0...— 0... cm 4 G N qiuAs oiogd SISG 8 I —v S (._.) sgseqiulis PII-Pd tau u! espenu' (v ca— Figure 12. 56 The stimulation of ALA-dependent PROTO accumula- tion by low concentrations of INH in the dark. Seedlings were incubated in 10 mM ALA with/ without various concentrations of INH for 24 hr in the dark. PROTO and PCHLD accumulation were assayed at the end of these time periods. The figure shows the effects of INH on tetrapyrrole accumulation after 24 hr of incubation. Control levels of PROTO and PCHLD were in nmol/g—fresh wt.: 66.9 t 6 and 140.9 i 16, respectively. 57 PCHLD ACCUMULATION .28... 7.2: cc. om ob ow ow . - a q - 1 <._< SEQ. E O O O Q uomqmu: x (H) O N NOIlV'InWDOOV OlOHd a O nonemwnsx O V O O S8 was inhibited only 10-17% compared to inhibitions of 38-50% for PCHLD. The differential inhibitory effect of INH on the synthesis of these tetrapyrroles may suggest a site of action somewhere between PROTO and PCHLD. Although this higher concentration of inhibitor may be acting in a different manner. The fact that 5 mM INH stimulated PROTO accumulation by 31% and had no effect on PCHLD seemed rather curious. If INH were blocking the conversion of PROTO to PCHLD, then we would have expected to see an inhibition of PCHLD (we will address this inconsistency in greater detail in the discussion). At a higher INH concentration (20 mM), however, PROTO was stimulated by 20% and PCHLD was inhibited by 20% - which is what we would have expected. The effect of INH on tetrapyrrole formation in low light was examined. When PROTO levels of ALA-fed barley shoots were estimated after exposure to low light in increasing concen- trations of INH, we noted a dramatic rise of 244% in the con- tent of PROTO in the INH-treated tissues as compared to controls (Figure 13). PCHLD synthesis remained refractory to INH over these same concentrations of inhibitor - the same effect observed in the dark. At higher concentrations, the inhibitory effects of INH on the synthesis of both tetra- pyrroles were displayed; PCHLD was inhibited maximally by 71% whereas PROTO was inhibited by only 32%. The formation of PCHLD appeared, therefore, to have a 2-fold greater sensitivity to INH than did PROTO, suggesting perhaps an Figure 13. 59 The stimulation of ALA-enhanced PROTO accumula- tion by low concentrations of INH in low light. Seedlings were incubated in 10 mM ALA with/ without various concentrations of INH for 8 hr in low light (3,000 ers/cmZ/sec). PROTO and PCHLD were assayed at the end of the 8 hr illumination period. 60 (H)NOI.I.V1anOOV CI'IHOd Ow ON Om <4<é _.._E o. .o 8585 e... e. ASE av :12: 8. cm oo 9. . . _ . . . _ .ts :00... m\_oE: :_ 2032:8330 ON 00 (H) NOILV'InwnOOV OlOHd 61 inhibitory site of action somewhere between these two tetrapyrroles. The inhibition of both PROTO and PCHLD at higher concentrations of INH may be due, however, to other general toxic effects. Further investigation into the effects of INH on pigment synthesis in shoots exposed to low light revealed that INH has no demonstrable effect on the synthesis of carotenoids (Table 2) - a process intimately associated with the forma- tion of the chlorOplast membrane (92). This in addition to other evidence argues against an explanation of INH's differ- ential mode of action on PROTO and PCHLD involving the inhibition of membrane formation. In the presence of added ALA, CHL synthesis was less sensitive to INH (50 mM) than PCHLD, an inhibition of 40% compared to 67% for PCHLD. This mitigates an inhibitory role for INH on membrane synthesis, since both PCHLD and CHL are presumably synthesized on mem- branes (102) and would have displayed similar extents of inhibition. We also demonstrated that the syntheses of PROTO, PCHLD, and CHL were differentially sensitive to INH (Table 2). The inhibitor strongly stimulated PROTO synthesis - as demonstrated earlier - between 28 and 66% for 5-20 mM INH. PCHLD remained unaffected and CHL slightly inhibited by 15% over these same concentrations. Increasing the INH concentration to 50 and 100 mM, resulted in differential inhibitions. The levels of PROTO and CHL synthesis were reduced by similar percentages: 32 and 44% for PROTO and 62 Table 2.--Effects of INH on the synthesis of tetrapyrroles and carotenoids in low light in the presence of ALA. Treatment % of Control concefififlation PROTO PCHLD CHL CAROTBNOIDS 0 100 t 10 100 t 12 100 i 12 100 t 16 5 mM 128 i 7 124 i 2 89 t 8 98 i 5 10 mM 131 t 4 96 i 3 87 i 8 102 i 8 20 mM 166 i 30 105 i 21 85 i 14 100 i 5 50 mM 68 i 1 33 i 16 60 i 1 98 i 16 100 mM 56 i 7 15 i 2 55 i 2 81 i 3 Etiolated shoots were incubated in 10 mM ALA + various con— centrations of INH (0-100 m) for 8 hr in low light (3,000 ergs/cmZ/sec). The levels of pigments were assayed at the end of this illumination period. Control levels of net synthesis for PROTO, PCHLD, CHL, and carotenoids in nmol/ g-fresh wt. were: 12.5 i 3, 23.0 i 3, 94.5 i 11, and 64.0 i 4, respectively. .— 63 40 to 45% for CHL. On the other hand, the Z-fold greater sensitivity of PCHLD synthesis to the hydrazide was again shown by its inhibition, ranging from 67 to 85%. These results raise the possibility that at higher concentrations (50—100 mM) INH may inhibit a step between PCHLD and CHL. To clarify whether there is indeed site of action of INH on the steps between the formation of PROTO and PCHLD, shoots were incubated in 5 mM ALA containing [4-14C] ALA in the dark with and without INH. The radiospecific activity (*SA) of the ALA in the incubation solution was .17 uCi/umol (300 cpm/nmol). After 3 hr incubation, the *SA's of the PROTO and PCHLD in the control tissue were 722 and 600 cpm/nmol respectively, or 30% and 25% of a theoretical *SA for tetra— l4c-ALA, of 2400 pyrroles, formed solely from the exogenous cpm/nmol (Table 4). [We are making the assumptions that all synthesis is derived from the unadulterated exogenous ALA (*SA= 300 cpm/nmol), and 8 molecules of ALA are used in forming one molecule of tetrapyrrole (75).] The *SA's of both PROTO and PCHLD were approximately one—quarter of the theoretical estimation, suggesting the presence of an endogen— ous pool of cold ALA or other intermediates leading to tetrapyrroles, which diluted the labelling by 75%. A differ- ential effect of INH on the amount of label incorporated into PROTO and PCHLD was clearly seen; the *SA of PROTO rose 52% over the control whereas the *SA of PCHLD remained constant - evidence supporting, the idea of a site of action 64 .s.e>.puemmce .m H N..m .e H m.mw .m 4 am .e A Nm no: Emcem-m\.oec c. 22. 62626.3 ece 26.: 22202 ece oeome .6 mameeecsm so: 62. .2: mN ceem< .Houecoe fizz.-. 6:. oe fizzH+U @opmonp-mzH cap mo onw base“ map 2H mm.9H>Huuw -ONcwh exp 20 mqmum new 09022 How 2HkuN2uoefiaozaw poxmmmm use wouzfio 6263 mamgwoume -oano ecu mo means paeEmNm Hmswfi>flwcfi one .voameamoumEOHno muomauxo paw .pouumuuxo .poumo>am: ohm: muoocm .2: IN was .mHH .m meefip :oeumnsocfi p< .NoE:\EmoomH ow vo>amn we: meeees.om eoeeeesuc. 2.6 c. <2< 62. mo Hm: mm: Hpu< UHOHueQmOprm .2268 one a. <2< e6. meoegm 26.662 a. 22202 was oeome mcaee.seeeee we mNuoenmeu .22 A ONI .w2 A meN .mv A vmm u2052\2520 :2 202wcoo 02H .2022266 02 6626022 we m we 2022m2zswum n uzc2NA-I .m2mecucxm we :oNu222222 :m "ccww H+VI m.2 uzefiumeuuv 2euuceu e22 ou ecumeeou mm acosume2u zone 23 262252;:2 mflmezuczm we w on» mucemoune» ceduflnflscw N .uceEumO2A we 2: m couwm ee=2Eheuee 6263 AIO was .22:02 .oeomm we memosucxm 622 can m_ on om2m eased A- Azzwv 22052662» 0: 2e n+ 2:22v aces -ume2u :22 .eo2hen 2: w esu 222236 :uon co A- .u:M22O mwucxuev 20 m+ ”22w22v uem\NEU\mw2eooom um coflumcflesamfi ou vemoaxe on 62:60 mmc2meeem 6;» 26:2 om ee2um> on @2506 22652802. uzm22 och .202cem 2; m 622 m222=e flucmfi2 v26 :zwv maceEu~e2u 26u2m 22:06 63 .2; I ch 6;» use 2; v um2 ecu ”maceEumeuu 2; I 2622:6326m 022 0222 weeN>2w we: 262202 2: m m2£H .v022en aceEume2u 2: w :m we emueou 0:» 222236 :22 25 ON A A2oE:\Eau O22v <2< 6622onm2-u<2 SE m we ceflue2em a new 0203 mm=NHVoem .o. A em.2 N A am me. A Nm.2 22 A NN em. A em.2 e A NN + + + + .e m2. A oo.2 m A .2 me. A 24.2 2 A .e ee. A eN.N N2 A om + - + + .w m2. 2 02.2 o A e. No. A NN.2 m A NN 22. A Ne.N A2 A NN . - - + .N N2. A e2.2 A A N. me. A 24.2 N A a A. A NN.2 A A m2 + - - - .e .2. A em.o 2 A .2 «Q. A mm.2 22 A 2N ee. A NN.2 m A NN A + - - .2 co. ASA wwm co. “co; muo no. “No.2 mwe- + + + - .v No. A 2e.e N A .2 co. A me.2 2 A eo2. no. A Nw.o N A e- - + + - .2 No. A 2e.2 N A Am 2e. A me.e m A 2N2- e A em.o 2 A a- - - + - .N 22. A ee.2 N A e No. A ee.. o. A e N2. A e.2 N A e . - 4 - .2 22uev mo2022>mmuuou we mwmezuzxm can ~0>2uuoam02 .vm A ONN 02m .mm A vom Awe A mmm .NOE:\EQU :2 2e2uceu 02H .A.2 u:052002uv 2022200 022 cu uc0sum022 nouwm 02eccxemtuou 2000 we 0n vasee .uLM22 0:0 HmUAEosu cues .muceeum02u 052u 202:3 mc22=0 .weoN20n 20Npe0=cem ozu oucfl 0002>N0 mm: u:0Eum0hu 2: w 0:» 020wen m< .:22 :5 ON A QEnmuuou we m2mezuczm 0:0 2 Hmm20>02 0gb--.m 0H309 71 opposite effect. Nicotinamide also mitigated the inhibition by INH of CHL synthesis (cf treatment 3 and 4); pre-treatment with nicotinamide reduced the inhibition of CHL synthesis from 35 to 11%. As a control, nicotinamide treatment had no effect on either the synthesis or *SA of any of the tetrapyrroles (treatment 2). We observed once again that light given before INH eliminates the inhibitory effects (treatment 8). These results suggest that nicotinamide acts in a manner dramati- cally opposite to that observed with INH - in a manner which antagonizes the effects of INH as does light. In an attempt to determine whether or not nicotinamide might possibly mitigate the inhibitory effect on ALA formation as well, we pre-treated shoots, as before, with 50 mM nicoti- namide. The tissue was then subjected to solutions of varying contents — 30 mM levulinate with and without 50 mM nicotina- mide with and without 20 mM INH. In addition, we varied the treatments during each 4 hr of the 8 hr illumination period (Table 6A). INH treatment for 4 hr gave the full inhibition of ALA formation (treatment 2). Nicotinamide did not reverse the effects of INH on ALA synthesis. Pre-treatment with nicotinamide was no different than pre—treatment with water in the extent of INH-inhibition (cf treatments 3 and 4); the reduction in inhibition appeared to be due to the light treatment. Exposing the tissue to 4 hr of light prior to INH- treatment reduced the inhibition of ALA synthesis from 53% to 13-14% (treatments 3, 4) - an effect observed in previous 72 .AH0>Huo0mm0A .m A Non .mo paw .v A 0H0 .0H achucoo 0:H .w0AAm> 0n fiasco wuq0Eu00Au pmgu om Am©0Ah0m A: A 03p ouqfl w0uA>Aw we: 00AA0Q 9:05u00pu A: w 0AA .AeoEAAomxo A0A2Am0 cA A< .022 25 om A 2moV zxm :5 om Ao mmA :5 om wcfiqflwpcoo m:0Hu5Hom ow p0hh0wmcmpu Cosy 0A0: mwaflfiw00m 0gb .thw 0gp GA mmoV AZXmV 0=Axowfigkm SE om go nmq SE om GA uc0Eum0HH “:0Ep00HuI0Hm .< .AAAA2 202 :2 :OAAAEAOA fluu0mm0u Am A m.Ao omw A.u3 nm0AmIM\HoE: :« 0A0 4mm paw 0H Houpcoo IA0p0p HH%:QOA0HAU How .>mq ZS om :H @0w0930sfl 0A0: mmmfiapomm .m:0au0:AEH0p0© g akmopmomHIN eflom oflcflaooflqomHv A A A.AN -omo 20A :umuvzzmzoo IA IAAAmmoge AAAAA:OAQ2 m A A.AA Azuou IA IAAAAAAAAAAIUAIA A A N.AA A A A.AA Azzmzou IA IAAAAAAAA AAUA UAaAAOUAeoA2 A A A.Am mozIzu IN IaAeAxoe2AIN-IcAeAAAA O2 A 0.0m 2 A A.AA :oou IA AAUA UAAAAoqu A A 2.mm A A a.ew :oou IA AAUA UAAAAOUA:OA2 m A A.Aw zoom Im.N erA UAeA2oeAso o A A.om zoom IA AAUA UAAA2ooAA Ame m0An Haksmopofiso 6:0 o-Nou :o :zH wo uoowmm--.ma oHLMH «a 88 an inhibitor of photorespiration. The photorespiratory inhibitors which had stimulated ALA synthesis were also examined for their effect on the respiration of [1-14C] glycine (Table 13). At 20 mM, a-HPMS inhibited this respira- tion by 34%, whereas 10 mM glycine hydroxamate effectively inhibited the respiration by 64%. Since both INH and glycine hydroxamate inhibited the evolution of 14CO2 to similar extents, it suggested that they might act on the same site of the photorespiratory pathway (59). D. The Effect of INH on the Synthesis of 6-Aminolevulinic in Anabaena variabilis The blue-green alga, Anabaena variabilis Kfitz, appears to synthesize ALA via the C-5 pathway (unpublished results, Avissar). Anabaena contains, therefore, a biosynthetic system for ALA formation similar to that of higher plant chloroplasts (103). Since INH has been shown to inhibit the in vivo syn- thesis of ALA in barley, we attempted to determine whether or 14C-1abelled not INH would inhibit the synthesis of ALA from precursors in Anabaena. Isonicotinic acid hydrazide was not only ineffectual in inhibiting the synthesis of ALA from [14C(U)] glutamate, but appeared on the other hand to stimu- late the incorporation of the labelled amino acid into ALA as determined by the method of Beale et al. (6) (Table 14). In the presence of 40 mM INH, the incorporation of labelled glutamate into ALA was stimulated by 30%. It was also apparent 89 Table 13.--Effect of A505, d-HPMS, and glycine hydroxamate on 14COZ-evolution. Treatment Respired l4C02(cpm/gm-FW) % Inhibition 1-14C-glycine 26,073 0 1-14C-glycine + 25 uM A505 18,090 30.6 + 20 mM a-HPMS 17,078 34.5 + 10 mM glycine 9,300 64.3 hydroxamate 3,4-14C-glutamate 10,226 0 + 25 uM A502 7,632 25.4 Two g of seedlings were illuminated at a light intensity of 3,000 ergs/cmZ/sec for 4 hr in beakers containing water. At the end of 4 hr, the seedlings were removed and the greenest 3 cm section of the seedling was excised and placed into 2 ml 14C- solutions containing .5 uCi/ml of l4C—glycine or 3,4- glutamate : inhibitor. The beakers and seedlings were placed into jars fitted with lids from which discs wetted with .1 M NaOH were suspended. The jars were returned to the light for 14 4 additional hours of illumination. The CO2 trapped on the discs were then measured by liquid scintillation counting. 90 .euowen mm necammes qu>HuoeoHpmc ecu pCm .veHmHucmsw .eHOCAxQ- :oaamHHHu:HUm vHDGHH m :H veumfin v:m museum: ecu mo Haez deuceo ecu Eouu ve>OEep mm: 2052 saw: venue: Megan hepHHu nepHow ezk .mq 2E om mcflcflmucou HHE NueEDHo> kuouv :oHusHom w =« xwmaw wusnhmz m :« veumnzucm mm: mcewnmc< we cadmcenmzm HHeu HE H < .o: mm cemmeumxe mH HuHmcem egu onen mm: <4< eHnmuueuev xHHmUHEezu we He>eH ex» eocwm n+0, ON + ewm VNmN mm A mmHm + + + HNH A «AHN wmo A 4mm.AH HA A mNmN - + + +o- mom moon - - eemtmusHmouex.uH=ge4H em omN Name - + + +0, NNH Hfimv - . eumcHeusm-uQH-v H A “ NH me A «HmH HmHH A momm + + + +0- AANH omoH + - + NH mmNH mmmm - + + : - - km- - +o mAHH ammm ecHu H UVH N HA vaqH omm.mH - + + a . . . ecHox m. - +o mam HNQ AH . H uqH H AHN a amQ.H~ mmaH H va.~qH mm A ooaq + + . +o, www.5H QNwH + - + AmNH A omH.mH mmaw A www.moH Am A mom“ - + + +o, an“ Nwmm - - mumsmuaHm.H:eevH Hos:\Equ =H eHoeesa-ms :5 em emcee tomtsueta eeHHean 6H6ce>a- mcemnmc< :H muomuauepa.uQ Ecud <4< mo mflmecucxmoHn ecu :0 :2H Ho uoeuwm-..vH eHan H 91 that, of all precursors tested, glutamate was preferentially converted to ALA - at least 6-times better than [14C(U)] a- ketoglutarate- the second best precursor, confirming the presence of the C-5 pathway for ALA synthesis in Anabaena. Neither [l- or 2—14C] glycine nor [1,4-14C] succinate were incorporated to any significant extents into ALA. Anabaena, therefore, did not appear to possess ALA synthase activity, suggesting that ALA was formed exclusively via the C—5 pathway from C-5 precursors. Although INH did not inhibit label incorporation from glutamate into ALA, it did have a dramatic effect on the incorporation of label from a-ketoglutarate into ALA. The hydrazide inhibited the conversion of d-KG to ALA by 89%. In addition, INH stimulated the respiratory release 14 of CO2 from [14C(U)] d-ketoglutarate by 25% whereas it had the opposite effect on the release of 14 C02 from [;4C(U)] glutamate, inhibiting the evolution by 37%. These results are not only consistent with a site of inhibitory action for INH between a-KG and glutamate, but also suggest that INH may act on ALA synthesis as an antagonist of the pyridoxal phosphate- dependent transamination of d—KG to glutamate. DISCUSSION The effects of INH on the biosynthetic pathway of chloro- phyll in greening barley shoots were examined. This study was undertaken to investigate the biosynthesis of ALA in higher plants. Isonicotinic acid hydrazide, a metabolic inhibitor reportedly an antagonist of pyridoxal phosphate (114), was intended to serve as a chemical probe for the presence of the pyridoxal phosphate requiring ALAS pathway for ALA formation in barley (cf 14). The existence of the C-5 pathway for ALA synthesis is well documented in a variety of greening organisms (5). The presence of the ALAS pathway in higher plants has also been reported, although less well documented than the C-5 pathway (85). The hydrazide of isonicotinic acid was chosen as a potential inhibitor of ALA formation primarily due to its antagonism of pyridoxal phosphate-dependent enzymes and, therefore, of ALAS (65, 14). However, during the course of these investigations, it became clear that INH had several sites of action in the biosynthetic pathway leading to chloro- phyll, some of which could not be related to an antagonism of pyridoxal phosphate-dependent enzymes. In order to eliminate any possible confusion concerning these multiple effects of INH, I will organize this discussion along the following lines: I. the effects of INH on tetrapyrrole 92 93 biosynthesis, II. the reversal of the INH-inhibition of tetrapyrrole biosynthesis by light, ALA, and nicotinamide, III. INH: mechanism of action against the biosynthesis of tetrapyrroles and ALA, and IV. additional modes of INH action. I. The Effects of INH on Tetrapyrrole Biosynthesis in Greening Barley Shoots INH appeared to exert at least two inhibitory effects on the greening process in barley: one on the synthesis of ALA, and another on the synthesis of chlorophyll. This was demon- strated by the different extents to which INH inhibited the syntheses of ALA and chlorophyll. The apparent inhibition constants for these inhibitions were 10 and 3 mM, respectively. The greater sensitivity of CHL to the inhibitor suggests that INH may act not only on the synthesis of ALA but also on a step in CHL biosynthesis subsequent to the formation of ALA. Had CHL synthesis been inhibited to the same extent as that of ALA, we could have ascribed both inhibitions to the action of INH on ALA synthesis. INH also appears to photosensitize tissue which has been fed ALA under low light — an effect not observed in tissue incubated in water or ALA alone. These observations suggested to us a site of action for INH on tetrapyrrole synthesis rather than on ALA formation alone. The accumulation of pigmented intermediates of the biosynthe- tic pathway leading to CHL are known to photosensitize plant tissue (11, 113). When barley was incubated in the dark in 94 ALA, to by—pass the effect of INH on ALA synthesis and, to examine the effect of INH on just that portion of the pathway from ALA to PCHLD, we saw a stimulation in the accumulation of PROTO and an inhibition of the accumulation of PCHLD (Figure 12). The stimulation of PROTO accumulation by INH was shown also to occur in the light and was consistent with the visual observation that the tissue appeared to have a slightly brownish color. The accumulated PROTO could account for the photosensitization of the INH-treated seedlings in low light (cf 113). It appears therefore that INH may act at a site between PROTO and PCHLD (Figure 16). If an inhibition is to occur between two points in the pathway of chlorophyll bio- synthesis, then we should expect to see an accumulation of that tetrapyrrole just before the blockage by INH. This is what we see when PROTO accumulates in the presence of INH. However, we should also see a decrease in the accumulation of the tetrapyrrole subsequent to the blockage - which we do not see. The formation of protochlorophyllide does not appear to inhibited at those concentrations of INH which stimulate the accumulation of PROTO. This inconsistency is puzzling. If, however, the formation of PCHLD is saturated due to the excess availability of ALA, then an INH block subsequent to PROTO synthesis may not necessarily result in an inhibition of PCHLD accumulation. This implies that the kinetic limitations on the synthesis of PROTO and PCHLD may not be the same. The synthesis of PROTO, for example, is a stromal process whereas Figure 16. 95 Pr0posed sites of action of INH on the biosynthetic pathway leading to chlorophyll. Site 1. ALA synthesis Site 2. Between protoporphyrin and protoch- lorophyllide 6 9 445 A De 10.111 i \’\/a‘l IZ_ m Geog .l .l.l3< EmmHLEzEij All! QALEESS AIII 3,2530 4| ESESHSE- was <3 :7; IE :7: _ .m E; .N E; .H E; o 130 (x. 131 responsible for glutamate synthesis and/or the glutamate—l— semialdehyde transaminase, and 2) a light-reversible antagonism of pyridine nucleotides inhibiting, perhaps, the dehydrogenase responsible for glutamate-l-semialdehyde formation. 6-Aminolevulinic Acid Synthase (ALSA): Does It Exist In Greening Barley Shoots?-—Could any of the INH effects on ALA synthesis be attributed to the presence of ALAS? In con- sideration of the evidence in the barley system that ALA is formed via the glutamate—l-semialdehyde pathway, it seems somewhat remote. The apparent pyridoxal phosphate-effect of INH involved in ALA synthesis seems to be due to either a direct interference of INH with the semi-aldehyde transaminase or the glutamate-forming transaminase and not with ALAS. It is clear from our labelling data in Anabaena that the *SA of the ALA formed from l4C-Z-glycine was .14% that from 14C(U)- glutamate (cf 22 cpm/nmol and 15,390 cpm/nmol). There is essentially no contribution of ALAS to ALA synthesis in blue- green algae. Whether this is equally true for barley shoots is not certain. Our experiments with A502 and INH (Table 9) may suggest that the C-5 pathway is the main pathway for ALA formation. We would expect the A505, an inhibitor of suc- cinate formation, would severely inhibit ALA synthase were it responsible for ALA formation in barley since succinate is a substrate for this enzyme (cf 70). We see, on the other hand, a stimulation in ALA synthesis with arsenite. 132 Stobart and Hendry (104) suggest that the small amount of label incorporated into ALA from [14C]—glycine is due to the levulinate method for ALA determination. At 30 mM levulinate, for example, the levels of glycine are elevated by 66% com- pared to controls, this suggests that any labelling by glycine of ALA could be diluted due to an increased glycine pool and therefore explain the low levels of label incorporation into ALA. The intracellular pools of glutamate and glycine in barley have been reported to be 670 and 140 nmol/g—fresh wt. (22). If the glycine pool is increased by 66%, there should relatively little effect on the *SA of glycine as compared to glutamate - since the amount of labelled precursor supplied in these comparative labelling experiments is the same for both amino acids. (In addition, the glycine level which they claim is l4-fold larger than that reported by Castelfranco and Jones, 22). Furthermore, levulinate treatment has no effect on the label incorporated into ALA from [14C]-glycine in Anabaena; controls without LEV incorporate the same amount of radioactivity as the LEV-treated (Table 14). Stobart and Hendry (47) suggest, moreover, that the ALAS pathway is operative in dark-grown leaves while the C-5 path- way is of quantitative importance in the light. They claim the incorporation of [14C]-glycine into ALA in the dark; however, they show neither their data nor an analysis of the labelling pattern determining the position of the radioacti- vity in the ALA carbon skeleton. In order to eliminate the possiblity that ALA is labelled non-specifically, it is 133 necessary to demonstrate that the label from [2-14C] glycine appears in C—5 of ALA, consistent with a labelling pattern that ALAS would give. If however the ALAS pathway does exist in dark—grown barley, it seems that it would have little significance for the plastids, since the levels of ALA are barely detectable in the dark. The light-inducible system for ALA formation and therefore chlorophyll synthesis appears to be the gluta- mate pathway. The presence of ALAS in barley remains equi- vocal, although the possibility exists that the incorporation of 14 C-glycine into the minute quantity of ALA observed in the dark is attributable to the synthesis of ALA used in hemo- protein formation in the mitochondria (cf 5). [It should be noted however that heme synthesis in rapidly greening barley has been suggested to be via the C-5 pathway (22).] IV. Additional Modes of Isonicotinic Acid Hydrazide Action A. INH as a Metal Chelator INH demonstrates a very strong affinity for heavy metals (1). INH, for example, chelates Cu2+ as in the Lowry assay (unpublished observation) and forms, when mixed with an equal molar solution of Cu2+, an INH-Cu2+ complex (3). In light of the fact, that chelators of iron - such as d,d'~dipyridyl and 8-hydroxyquinoline - induce the synthesis of Mg-porphyrins in etiolated beans and barley (95, 25, 40), it seems rather 134 important to consider the effects of INH on tetrapyrroles as a consequence of metal chelation. The most notable effect on tissue treated with iron chelators is the large accumulation of MgPROTO ME (25, 95). INH-treated barley seedlings do not appear to accumulate MgPROTO ME. Moreover, dipyridyl stimu— lates both the biosynthesis of Mg—porphyrins, in the absence of ALA, and the biosynthesis of ALA itself in beans; we observe neither effect in barley. These chelators act to inhibit heme synthesis, thereby removing a reputed feedback inhibition by heme on the rate-limiting step(s) of tetra— pyrrole formation (25), thus deregulating the synthesis of ALA and Mg~tetrapyrroles. We observe, however, the opposite effects with INH: inhibitions of both ALA and PCHLD. If INH were acting as an iron Chelator, then we should not observe the reversals of INH effects with light or nicotinamide treatments. Other porphyrinogenic compounds like pyridine- Z-aldoximine, pyridine-Z—aldehyde, and picolinic acid do not display the inhibitory profile of INH. This suggests that the modes of action of INH, which we see, are not due to any intrinsic potential to chelate metals. B. INH as a Photorespiratory Inhibitor of the Glycolate Pathway INH inhibits the photorespiratory conversion of glycine to serine in Chlorella and in leaves of higher plants (87, 59, 99). This conversion is catalyzed by the combined action of glycine decarboxylase and serine hydroxymethyltransferase 135 in the mitochondrion (59). Apparently, INH affects the pyridoxal phosphate-dependent decarboxylation reaction responsible for the photorespiratory evolution of CO2 in higher plants (59). In order to determine whether the effects of INH which we see were due to an inhibitory effect on photo- respiration, we screened several chemical inhibitors - known to block the glycolate pathway - for their ability to inhibit ALA and chlorophyll synthesis. The two inhibitors (10 m): glycine hydoxamate, a glycine analog which presumably blocks the conversion of glycine to serine, and u-HPMS, an inhibitor of glycolate oxidase, stimulate ALA synthesis by llOand 52%, respectively (Table 11). This is quite inconsistent with the effect we obtain with INH. Additional pieces of data do not support a role of INH‘s action on ALA and CHL as a consequence of its effect on the photorespiratory pathway: 1) glycine, which accumulates in the presence of INH (87, 99), stimulates ALA formation by 26% at 20 mM (Table 11), 2) INH is slightly inhibitory of the glutamate-glyoxylate aminotransferase (87), which, according to Zelitch (123, 71), causes an accumulation of glutamate. Glutamate should, at least, stimulate ALA formation, and 3) unlike INH, both glycine hydroxamate and d-HPMS* do not inhibit CHL formation (Table 11). From these *Hendry and Stobart (48) have found, to the contrary, that 10 mM d—HPMS inhibits CHL synthesis by 39%. Their illumination period was 22 hr compared to 8 hr for our ex— periments. d—HPMS is known to be unstable after exposures of light longer than several minutes (Tolbert, personal communi- cation). It is possible that their effects are a consequence of the relase of the sulfonic acid group upon light-stimulated degradation of d-HPMS). 136 observations, it seems apparent that INH does not act on ALA and CHL formation in the same manner as any of the inhibitors of the glycolate pathway. The apparent stimulations of ALA synthesis by glycine hydroxamate and d-HPMS may be due to an inhibition of carbon flow through the glycolate pathway, which may compete indirectly with the biosynthetic pathway of CHL. Blocking glycolate oxidation with u-HPMS, for example, inhi- bits photorespiration in leaf discs from tobacco and increases net photosynthesis by 50% (84). Perhaps, this accounts for the increased ALA formation. Glycine hydroxamide may stimu— late glutamate accumulation, since an inhibition of the gly- cine to serine conversion results in the accumulation of glyoxylate and glutamate (cf 84). In addition, we have shown that INH is fully capable of acting as an inhibitor of the mitochondrial conversion of [l-14 C]-glycine to serine, in- hibiting by 83% (Table 12). Although INH clearly inhibits the photorespiratory glycolate pathway, its effects on ALA and tetrapyrrole synthesis do not appear to be a manifestation of this particular site of action. V. Summary In overview, we have seen that INH does not act with one specific mode of action on the pathway of chlorophyll bio- synthesis. Isonicotinic acid hydrazide proved therefore to be a non-specific inhibitor, and not just the pyridoxal- phosphate antagonist for which it was originally intended. 137 The hydrazide appears to have several inhibitory sites in the tetrapyrole pathway. There seems to be a site of action on ALA synthesis and a site on tetrapyrrole synthesis, pre— sumably between the synthesis of PROTO and PCHLD. The partial reversal of chlorophyll synthesis by ALA supports a direct effect of INH on ALA synthesis. The accumulation of PROTO and large increase in the *SA of this porphyrin With INH treatment in [14C]ALA-fed tissue support a site for INH action after PROTO formation. Both light and nicotinamide pre- treatment mitigate the inhibitory effects of INH on tetra- pyrroles. We have offered a hypothesis for the INH effects on tetrapyrroles formation involving a perturtation of the pyridine nucleotide pool. In contrast, the hydrazide appears to act differently on ALA synthesis. In this case, INH may act like a pyridoxal phosphate antagonist. In conclusion, INH has offered a means to examine the sensitivity of the chlorophyll biosynthetic pathway to an inhibitor with several modes of action. Its apparent involve- ment in antagonizing both pyridoxal phosphate - requiring reactions and the pyridine nucleotide pool has given us some insight on the regulation of the biosynthesis of tetrapyrroles in greening barley. 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APPENDIX APPENDIX Hutner's Nutrient Medium Hutner's stock (—Mg2+): 59 X normal strength Solution A: in ~400 ml glass distilled water, chemical in grams Ca(NO - 35.4 3)2 EDTA(free acid)* - 50.0 KZHPO4 - 40.0 KOH (85% pellets) 24-26 NH4NO3 — 20.0 * EDTA not very soluble unless under alkaline conditions Solution B: in ~300 ml glass distilled water ZnSO4'7H20 - 6.59 H3B03 - 1.42 NaZM04-2HZO- 2.52 CuSO °5H O - 0.394 2 2 + 1 N HCl until cloudiness disappears Solution C: ~100 ml of water FeSO °7H O - 2.49 4 2 Add solution A and B, adjust pH with l N HCl, then add solution C and bring up to correct volume. Mg:+Stock: 25 gm MgSO4-7HZO/liter of glass distilled wa er Hutner's Nutrient Solution: 25 ml of Hutner's stock + 25 ml MgZ+ stock and bring volume up to 2% liter with water. 150 WICHIGQN STQTE UNIV. LIBRRRIES WlWIWWI""INIWIWIWIIHHIHMWWHI 312931®0634199