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H b S in prevalence of abnormal hemoglobins. The p r o p o r t i o n of H b E in heterozygotes decreases markedly in individuals who also have t~-thalassemia. This p h e n o m e n o n is analogous to that encountered in AS heterozygotes described above, and probably has the same explanation 2s. Impaired affinity of/3E chains for o~ chains might also explain the unusual severity of the clinical s y m p t o m s in individuals who are doubly heterozygous for H b E and fl-thalassemia. The distribution of Hb A2(a2~52) in red cells has often puzzled hematologists. Hb A2 comprises about 2% of the total hemoglobin in normal red cells. In individuals with the fl-thalassemia trait (an impairment of/3 A chain synthesis) Hb A2 is about twice normal. In contrast, Hb A2 is s u b n o r m a l in a-thalassemia trait (deletion of two a chain genes) as well as in patients with iron deficiency and sideroblastic anemia ~. These two acquired disorders also evince deficient a chain synthesis. The decreased levels of H b A2 in all three conditions may be explained by a relatively low affinity of ~5chains for o~ chains, compared to that of flA chains for a chains. Recently Lee et aL so have presented some experimental evidence that s u p p o r t this hypothesis. Interestingly, individuals with both h o m o z y g o u s sickle cell anemia and cz-thalassemia (2 gene deletion) have normal or elevated Hb A2, presumably because the ¢5 chains can compete effectively with fls chains for the limiting a m o u n t s of a chains. Differential affinity of normal and variant subunits for partner subunits may account for occasional unusual hemoglobin patterns. For example, heterozygotes with Hb Hacettepe (a~fl2 ~2~oj.~;lu) have significantly more of the variant hemoglobin than Hb A in their red cells. The abnormal residue is located at the a~flt interface, a region of closest contact between ct and/3 chains in the a2f12 t e t r a m e P k Therefore a substitution at this site might have a marked effect on the rate of formation of the o~fl dimer. In fact, Rucknagel and his associates s2 have found that this variant has supranormal stability. It is likely that the rate at which a chains and flmc~t~,~ chains react to form the aft dimer exceeds that for a and/3A chains. Thus, the phenotypes of a n u m b e r of h u m a n hemoglobin disorders can be explained by abnormalities in the assembly of subunits to form the functioning hemoglobin tetramer. Similar abnormalities may apply to a larger group of congenital and acquired disorders involving multi-subunit proteins.

Acknowledgements The authors' work was supported by National Institutes of Health grants H L 16927 and H L 24021.

References 1 Bucci, E. and Fronticelli, C. (1965)J. Biol. Chem. 240, PC551-PC552 2 Antonini, E., Bucci, E., Fronticelli, C., Chiancone, E., Wyman, J. and Rossi-Fanelli, A. (1966) J. Mol. Biol. 17, 29-46 3 Tainsky, M. and Edelstein, S. J. (1973)J. Mol. Biol. 75,735-739 4 Kazim, A. L. and Atassi, M. A. (1980)Biochem. J. 185,285-287 5 McGovern, P., Reisberg, P. and OIson, J. S. (1976)J. Biol. Chem. 25I, 7871-7879 6 Rollema, H. S., Gros, G. and Bauer, C. (1980) J. Biol. Chem. 255, 2756-2760 7 Friedman, F. K. and Beyehok, S. (1979) Ann. Rev. Biochem. 48, 217-250 8 McDonald, M. J. and Noble, R. W. (1972)J. Biol. Chem. 247, 4282-4287 9 Geraci, G., Parkhurst, L. J. and Gibson, Q. H. (1969) J. Biol. Chem. 244, 4664-4667 10 Salahuddin, A. and Bucci, E. (1976) Biochemistry 15, 3399-3405 11 McDonald, M. J., Turci, S. M. and Bunn, H. F. (1980)Fed. Proc. 39, 1888 12 Sugita, Y. (1975)J. Biol. Chem. 250, 1251-1256 13 Shaeffer, J. R., Kingston, R. E., McDonald, M. J. and Bunn, H. F. (1978) Nature (London) 276, 631-633 14 Shaeffer, J. R. (198(I) J. Biol. Chem. 255, 2322-2324 15 Neel, J. V., Wells, I. C. and Itann, H. A. ( 1951 ) J. Clin. Invest. 30, 1120-1124

16 Huisman, T. H. J. (197 7) Hemoglobin 1,349-382 17 Steinberg, M. H., Adams, J. G., tit and Dreiling, B. J. (1975) Br. J. HaematoL 30, 31-37 18 Lehmann, H. (1970) Lancet 2, 78-80 19 Dozy, A. M., Kan, Y. W., Embury, S. H., Mentzer, W. C., Wang, W. C., Lubin, B., Davis, J. R., Jr. and Koenig, H. M. (1979) Nature (London) 280, 605--607 20 Brinenham, G., Lozoff, B., Harris, J. W., Kan, Y. W., Dozy, A. M. and Nayudu, N. V. S. (1980) Blood 55,706-708 21 Weatherall, D. J. (1964) Ann. N.Y. Acad. Sct. 119, 463-473 22 Abraham, E. C. and Huisman, T. H. J. (1977) Hemoglobin 1,861-873 23 DeSimone, J., Kleve. L., Longley, M. A. and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 57,248-254 24 Shaeffer, J. R., Kleve, L. J. and DeSimone, J. (1976) Br. J. Haematol. 32, 365-372 25 Boyer, S. H., Hathaway, P. and Garrick, M. D. (1964) Cold Spring Harbor Syrup. Quant. Biol. 29, 333-346 26 Bank, A., O'Donnelk J. V. and Braverman, A. S. (1970)J. Lab. Clin. Med. 76, 616-621 27 Kantor, J. A., Turner, P. H. and Neinhuis, A. W. (1980) Cell 2l, 149-157 28 Tuchinda, S., Beale, D. and Lehmann, H. (1967) Humangenetik 3, 312-3 l 8 29 McCormack. M. K. (1980) Clin. Chim. Acta 105, 387-391 30 Lee, T. C. K., Graves, G. D., Nerurkar, S. G. and Kim, B. C. (1978)Fed. Proc. 37, 1390 31 Perutz, M. F. and Lehmann, H. (1968) Nature (London) 219. 902-909 32 Winter, W. P., Rucknagek D. L. and Whinen, C. F. (1977) Clin. Res. 25,325A

Ethylene the gaseous plant hormone: mechanism and regulation of biosynthesis Douglas O. Adams and Shang Fa Yang Ethylene is a natural regulator o f plant growth and d e v e l o p m e n t and has been used, wittingly or unwittingly as a fruit ripening agent for m a n y years. Its path o f synthesis has recently been elucidated and there is n o w s o m e understanding o f h o w ethylene production is regulated.

Ethylene is the simplest organic c o m p o u n d which is biologically active in trace amounts; it is a natural product of plants, and its effects are spectacular and commercially importantk The early use of smoke and incense as ripening agents can be attributed to their ethylene content. Following the demonstration that ethylene was a plant h o r m o n e initiating fruit ripening, many other plant processes, ranging from germination to senescence, were recognized as being regulated by ethylene x. Recent elucidation of the pathway of Douglas O. Adam.~ and Shang Fa Yang are at the Department of Vegetable Crops, University of California, Davis, California, 95616, U.S.A.

ethylene biosynthesis has led to important information about how production of this important plant h o r m o n e is regulated.

S-adenosylmethionine as an intermediate in ethylene biosynthesis Methionine was first suggested as a possible precursor of ethylene by Lieberman and Mapson 2 as it was rapidly converted into ethylene in a model system consisting of Cu 2+ and ascorbic acid. It is now believed that methionine serves as the biological precursor of ethylene in all higher plant tissues 3. In the conversion of methionine to ethylene, C - 1 is converted to CO2, C - 2 to formic acid and C - 3 , 4 to ~, Elsevier/North-HollandBiomedicalPress1981

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ethylene. However, the sulfur atom is retained in the tissue ~ and this is biologically important as the very low concentration of methionine in plant tissues would become limiting if sulfur were not recycled. A d a m s and Yang 4 examined the fate of the C H 3 S - group of methionine during its conversion to ethylene in apple tissue and discovered that it is converted into methylthioadenosine and its hydrolysis product, methylthioribose, both of which are known metabolites of S-adenosylmethionine (SAM). As predicted, methylthioadenosine was effectively cycled back to reform methionine via methylthioribose 4. These data suggest that in the sequence of ethylene biosynthesis from methionine, SAM serves as an intermediate. I-Aminocyclopropanecarboxylic acid as an intermediate in ethylene biosynthesis In an effort to identify the intermediate linking SAM and ethylene, A d a m s and Yang 5 compared the metabolism of methionine in apple tissue in air and in a nitrogen atmosphere. In air, methionine was efficiently converted to ethylene. In nitrogen, however, it was not metabolized to ethylene but was converted to methylthioribose and a compound later identified as 1-aminocyclopropane-Icarboxylic acid (ACC). In the presence of air A C C was rapidly converted to ethylene, indicating that A C C is an intermediate and that the conversion of A C C to ethylene is CH3-S - CH2-GH2- CH- C00-4{

(MET}

Ribose

ATP

C4 - Acceptor

PPi + Pi + CH3-S - CH2- CH2- CH-COO-

I

I

CH3- S-Rlbose

+

~ ' - - ~ Ado

NH3

Ado

+ H2C

NH3

H~,C

COO(ACC)

O~,- - ~ < ~

CH2

:

f

Anaerobios=s

Uncouplers CO2+

CH2

Temp > 35 ° C Light

Fig. 1. Regulation of ethylene bio,~ynthesis WZr/A: thi,s reaction is normally suppressed and is the rate-limiting step in the pathway; liP': induction of.synthesis of the enzyme; ~ ~inhibition of the reaction. Met, A do and Ado, A VG and A OA stand for methionine, adenosine and adenine, aminoethoxyvinyg(vcine, and arainoo'(yacetic acid, re.~pectively.

oxygen-dependent. These data suggest the following sequence for the pathway of ethylene biosynthesis: methionine --~ SAM --~ A C C -*ethylene. Adams and Yang also observed that aminoethoxyvinylglycine, a potent inhibitor that blocks conversion of methionine to ethylene 6, did not block the conversion of methionine to Sadenosylmethionine or the conversion of A C C to ethylene; however it did effectively block the conversion of methionine to ACC. Thus, aminoethoxyvinyl-glycine must exert its inhibitory effect by blocking the conversion of SAM to ACC. Independently, Lfirssen and co-workers observed that application of A C C enhanced ethylene production in a n u m b e r of plant species. They likewise found that ACCdependent ethylene production was completely inhibited under anaerobic conditions and was insensitive to inhibition by aminoethoxyvinyglycine. Although the intermediate role of A C C in ethylene biosynthesis was not shown, Lfirssen et al., proposed that A C C was derived from SAM and served as an ethylene precursor. Production of ACC from SAM: ACC synthase Soon after the pathway for ethylene biosynthesis became known, Boiler et al. 8, reported that cell-free extracts prepared from tomato fruit were capable of converting SAM to ACC. They reported that the enzyme was soluble and strongly inhibited by aminoethoxyvinylglyrine as predicted by A d a m s and Yang. The Km for SAM was estimated to be 13 p,M and the enzyme specifically utilized SAM as substrate. Employing labeled SAM, Yu et al. 9, confirmed that SAM was converted to A C C and methylthioadenosine by the enzyme preparation from tomato fruit. In addition they showed that S-adenosylethionine could serve as a substrate for A C C synthase although it was less efficient as a precursor than SAM. They 9 also demonstrated that low concet, trations of pyridoxal-phosphate could activate A C C synthase and that the enzyme was strongly inhibited by aminooxyacetic acid, another well-known inhibitor of enzyme reactions dependent on pyridoxal-phosphate, and by aminoethoxyvinylglyrine. These observations strongly support the view that A C C synthase is a pyridoxal enzyme, but final proof awaits purification of the enzyme. The involvement of pyridoxal phosphate in the conversion of methionine to ethylene was first suggested by Owens et al. lo. They discovered that rhizobitoxine, an analog of aminoethoxyvinylglyrine and a known inhibitor of pyridoxal enzymes, could inhibit conversion of methionine to ethylene.

Later canaline, another known inhibitor of enzymes dependent on pyridoxalphosphate, was also shown to be an inhibitor of ethylene biosynthesisn, reinforcing the idea that pyridoxal phosphate was involved in one of the biochemical reactions leading to ethylene. It is now clear that these pyridoxat phosphate inhibitors inhibit ethylene biosynthesis by blocking the conversion of SAM to ACC. The conversion of SAM to methythioadenosine and a cyclopropane compound, A C C is a typical y-elimination (l,3-elimination) reaction. Organic chemists have shown that T-eliminations proceed readily involving an intermediate carbanion as depicted below: -X

X

)Y

+

it is known that pyridoxal enzyme (Py.E) facilitates elimination o f the proton from the a-carbon o f an amino acid yielding a carbanion TM. Since the positive sulfonium group o f S A M is an excellent leaving group, once the carbanion is formed, an intramolecular nucleophilic displaeement reaction can occur, resulting in the elimination of methylthioadenosine and the formation o f ACC. Py-E + + CH3"S'CH21CH2"CHi COOH~ CH3 S CH2 CHz CH COOH~ Ado NH2 Ado N: Py. E

H+

PyE MTA H2C

f- ~CH2-CCOOH _Z~ c% +' s )CH~ £*do

N=Py,E

\ / COOH L I/C\

H2C

N=Py.E

H2C

\ / COO~ I/C\

H2C

NH2

Conversion of ACC to ethylene Although the enzyme system catalyzing the conversion of A C C to ethylene appears to be present in most plant tissues, a natural cell-flee system capable of converting A C C to ethylene has not been demonstrated. The characteristics of the conversion A C C to ethylene in v i v o suggest that this enzyme is membrane-associated, labile and disrupted by various treatments TM. Nevertheless, it is clear from the in v i v o data that the reaction is oxygendependenP; furthermore it is inhibited by Co=+ ~4, temperatures above 35°C '3, lighP ~ and uncouplers such as dinitrophenoW. Until the enzyme system responsible for the conversion of A C C to ethylene is isolated and characterized, it is perhaps premature to discuss its biochemical reaction. Nevertheless it is interesting to note that the chemical oxidation of substituted cyclopropylamine to ethylene via a nitrenium ion intermediate has been documented ~6. Analogous to the chemical oxidation, A C C could be oxidized

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T I B S - J u n e 1981

enzymatically either by hydroxylation followed by dehydration, or by dehydrogenation of the amino group yielding a nitrenium intermediate which could then fragment spontaneously into ethylene and other products:

L~

[o] COOH

o.- [~..~<:~_. ~ ~/Y

COOH

cN CH2

COOH

Induction of ethylene production by auxin, flooding, and wounding With the elucidation of the ethylene biosynthetic pathway our understanding of the regulation of ethylene production in some physiological processes has been greatly advanced. One important observation that emerged immediately was that the system that converts ACC to ethylene appears to be constitutive in most plant tissues with the notable exception of preclimacteric (unripe) fruit tissue. This conclusion is based on the observation that ACC applied to a wide variety of plant tissues including roots, stems, leaves, inflorescences and fruit, causes an increase in ethylene production from 10-1000 times the control value ~7. The natural plant hormone indoleacetic acid (known as IAA or auxin) induces ethylene production in a number of plant tissues and many of the effects of auxin on plant growth are now attributed to auxininduced ethylene productionL The mechanism of auxin-induced ethylene production was unclear until recently. Yu et al.~8, found that ethylene production from mung bean hypocotyls was greatly increased by treatment with ACC even without auxin present and that ACCdependent ethylene production was insensitive to aminoethoxyvinylglycine whereas auxin induced ethylene production was completely blocked by aminoethoxyvinylglycine. Noting that the conversion of methionine to SAM did not require auxin induction, and that the system for converting ACC to ethylene was constitutive, they concluded that auxin stimulated ethylene production by inducing ACC synthase and that this step was rate limiting for ethylene production. Further support for this hypothesis was provided by Yu and Yang ~' and Jones and Kende TM. They demonstrated that the level of endogenous ACC closely paralleled ethylene evolution from mung bean or pea stem sections treated with different concentrations of auxin and that the activity of ACC synthase was higher in tissue incubated with auxin than that incubated in water only. These observations support the conclusion that auxin stimulates ethylene production by increasing the activity of ACC synthase.

Early work had shown that waterlogging of some plants can lead to petiole epinasty (more rapid growth in the upper side resulting in bending the petiole downward), adventitious rooting and reduced growth. Later work indicated that induced epinasty by flooding resulted from increased ethylene concentrations in the shoots of flooded plants 2°. It had been proposed that in response to flooding conditions a 'signal' is synthesized in the anaerobic root and transported to the shoot where it causes ethylene evolution. However, the nature of the "signal' was unknown. Bradford and Yang 21 have recently shown that ACC is synthesized in the anaerobic root in response to flooding and subsequently transported through the xylem to the shoot where it is aerobically converted to ethylene, giving rise to epinasty of the petioles. ACC is therefore the 'signal'. Since the rate of ethylene evolution by roots of non-flooded plants was too low to account for the increased ethylene production by shoots of the flooded plants, Bradford and Yang concluded that anaerobic stress not only blocks conversion of ACC to ethylene, but also increases ACC synthesis in the roots by stimulating or inducing ACC synthase. Induction of ACC synthase seems to be the mechanism whereby many of the stimuli known to increase ethylene production act. Wounding is well known to induce a rapid increase in ethylene production and this ethylene is known to be derived from methionine 1. Yu and Yang 22 have reexamined the biosynthesis of wound ethylene in light of the new information about the pathway. They studied woundinduced ethylene in mung bean hypocotyls, peel tissue of orange and the tissue of unripe green tomatoes. They found that the increase in wound ethylene was preceded by an increase in ACC in the wounded tissue whereas the level of SAM was unchanged. In tomato tissue they found that the increase in ACC was coincident with an increase in the amount of ACC-synthase activity. Treatment of the tissue with cycloheximide blocked woundinduced ethylene production, accumulation of ACC and development of ACC synthase activity. This work suggests that wounding induces the formation of ACC synthase - the rate controlling enzyme in the pathway. The picture emerging from the recent work on the pathway of ethylene biosynthesis in higher plants (Fig. 1) points out the importance of ACC synthase in controlling ethylene production at the level of induction of new protein synthesis. This enzyme appears to be the rate limiting step

in most of the tissues that have been examined. Therefore, one might speculate that many of the stimuli known to cause ethylene production in higher plant tissue will be mediated by increasing ACC synthase. Understanding how such a diverse set of signals such as auxin treatment, flooding of roots, and wounding can all induce the same key enzyme, ACC synthase, is a challenging problem for future work.

ACC in plant biology: where to now? Before the recent interest in this unusual amino acid sparked by its recognition as an ethylene precursor, ACC was for the most part ignored. It was simply one of the many so called non-protein amino acids that have been isolated from plants. Burroughs 2a identified this cyclopropane amino acid in Perry pears and cider apples over 20 years ago and Vahatalo and Virtanen 24 demonstrated that it was also present in the ripe fruits of the cowberry, Vaccinium vitisidaea. The study of this amino acid has been made considerably easier with the .recent development of a rapid, simple and sensitive method for the determination of ACC in plant extracts 25. The amount of ACC in an amino acid extract can be determined by degrading ACC to ethylene in the presence of NaOCI and Hg2+; the ethylene thus liberated is then determined by gas chromatography. The method was shown to be very specific and with the aid of a suitably sensitive gas chromatograph one can detect as little as 5 picomoles of ACC. We now have a better understanding of how ethylene is produced in higher plants. Methods for assaying ACC (the ethylene precursor), and ACC synthase (the enzyme responsible for ACC formation), are available. ,~lthough these tools have only been in use for a short time, many important physiological questions concerning the regulation of ethylene biosynthesis have begun to be answered. The prospects for a more complete understanding of how plants control ethylene production seem good indeed.

Acknowledgements Our work cited herein was supported by research grants (PCM75-14444 and PCM 78-09278) from the National Science Foundation.

References 1 Abeles, F. G. (1973) Ethylene in Phmt Biology, AcademicPress,New York 2 Lieberman,M. and Mapson,L W. (1964) Nature (London) 204, 343 345 3 Yang, S. F. (1974) Recent Adv. Phytoehem. 7, 131 164

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4 Adams, D. O. and Yang, S. F. (1977) Plant Physiol. 60, 892-896 5 Adams, D. O. and Yang, S. F. (1979) Proc. Natl. Acad. Sci. U.S.A. 76. 170-174 6 Lieberman, M., Kunishi, A. T. and Owens, L. D. (1975) in Facteurs Et Regulation De La Maturation Des Fruits Colloques lnternationaux Du Centre National De La Recherche Scientifique No. 238, pp. 1-9, Centre National De La Recherche Scientifique, Paris 7 LiJrssen, K., Nauman, K. and Schroder, R. (1979) Z. Pflanzenphysiol. 92,285-294 8 Boiler, T., Herner. R C. and Kende, H. (1979) Planta 145,293-31/3 9 Yu, Y. B., Adams, D. O. and Yang, S. F. 11979) Arch. Biochem. Biophys. 198, 280-286 10 Owens, L. D., Lieberman, M. and Kunishi, A.

( 1971) Plant Physiol. 48, 1-4 l 1 Murr, D. P. and Yang,S. F. (1975) Plant Physiol. 55, 79-82 12 Davis. L. and Metzler, D. E. (19721 in The Enzymes (P. D. Boyer, ed.) vol. 7. pp. 33-74, Academic Press,New York 13 Yu, Y. B., Adams, D. O. and Yang,S. F. (19811) Plant Physiol. 66, 286-290 14 Yu, Y. B. and Yang, S. F. (1979) Plant Physiol. 64, 1074-1077 15 Gepstein, S. and Thimann, K. V. (1980) Planta 149, 191",199 16 Hiyama, T., Koide, H. and Nozaki. H. (1975) Bull. Chem. Soc. Japan 48, 2918-2921 17 Cameron. A. C., Fenton, C. A. L., Yu. Y. B., Adams, D. O. and Yang, S. F. (1979) Hort. Science 14. 178-180

18 Yu, Y. B.. Adams, D. O. and Yang, S. F. (1979) Plant Physiol. 63, 589-590 19 Jones, J. F. and Kende, H. 119791 Planta 146, 649-656 211 Jackson, M. B. and Campbell, D. J. (1976) New Phytol. 76, 21-29 21 Bradford, K. J. and Yang, S. F. (1980) Plant Physiol. 65,322-326 22 Yu, Y. B. and Yang, S. F. 11981}) Plant Physiol. 66, 281 285 23 Burroughs, L. F. 11957) Nature (London) 179, 3611-361 24 Vahatalo, M. L. and Virtancn, A. 1. 11957)Acta Chem. Seand. 11. 741-743 25 Lizada, M. C. C. and Yang, S. F. (1979)Analvt. Biochem. 11)0, 140-145

50 Years Ago van Niel and the unity of photosynthesis Harmke Kamminga Ever since the classic studies of Priestley, Ingenhousz and Senebier in the 18th century, it has been known that photosynthesis in green plants involves the assimilation of carbon dioxide and the evolution of oxygen. The relation between these two phenomena, however, long remained a subject of uncertainty, especially after the discovery of photosynthetic bacteria that assimilate CO2 without releasing O~. This issue was finally clarified by Cornelis van Niel, 50 years ago ~, in a proposal for a unified mechanism of photosynthesis that laid the foundations for modern thinking on the subject. In 1931, van Niel suggested that all photosynthetic processes involve a series of co-ordinated oxidations and reductions by means of hydrogen transfer and that the final oxidation product is 02 only if water acts as the primary hydrogen donor. The overall reaction could be expressed as follows: light

CO2 + 2AH2 --+ (CH20) + H20 + 2A where AH2 is a suitable hydrogen donor and (CH20) represents organic products such as sugars, van Niel subsequently made explicit the idea that the overall reaction consists of two basic steps: (i) the photochemical splitting of a hydrogen donor into a reducing and an oxidizing entity, and (ii) a series of co-ordinated, light-independent oxidations and reductions 2. Harmke Kamminga is an associate of the Department of History and Philosophy of Science, CheLsea College, University of London. ~lailing address: 178 Sturton Street, Cambridge CBI 2QF, U.K. klsevier/North-Holland BiomedicalPress 1981

The key to van Niel's scheme was provided by the green and purple sulphur bacteria, which he studied extensively, first in Delft and later in Pacific Grove, California. His investigations suggested that light-dependent CO2 assimilation in R h o d o b a c t e r i a , C h r o m a t i u m and other pigmented sulphur bacteria involves the reduction of CO~ by hydrogen, balanced by the oxidation of inorganic substrates such as H2S, S or H2SO3. For example, bacteria that use H2S as substrate effect the following transformation: light CO2 + H2S ~ o r g a m c matter + S van Niel found that, in general terms, bacterial photosynthesis appears to obey the general equation given earlier. The stoichiometry of specific cases was tentatively confirmed by chemical analysis, van Niel also saw that his scheme could account for plant photosynthesis on the assumption that water is the primary hydrogen donor in this case. Thus, a unified picture of photosynthesis was obtained and one that is, in its general outline, consistent with modern theory. The idea that photosynthesis involves a co-ordinated, but in principle, independent reduction of CO2 and oxidation of a hydrogen donor received experimental support in the 1930s and early 1940s. R. Hill showed that illuminated suspensions of chloroplasts that did not assimilate CO2 still evolved O= in the presence of a suitable oxidizing agenP. This oxidizing agent, potassium ferric oxalate (one of several agents now known as Hill oxidants), was

reduced in the process, just as CO2 is normally. Conversely, Gaffron demonstrated that 02 release could be eliminated from the photosynthetic process in S c e n e d e s m u s and related algae that could be adapted to using hydrogen gas instead of water as hydrogen donor 4. In addition, the radioisotope studies of Ruben and Kamen and colleagues supported the implication of van Niel's scheme that the 02 released in plant photosynthesis is derived from H20, and not from CO2 *s. Besides stimulating much research, van Niel's hypothesis had the merit of being able to accommodate a number of observations that were not easily reconciled on previous interpretations of photosynthesis. According to the dominant 19th-century view of photosynthesis, CO2 assimilation and 02 evolution were necessarily connected: CO2 was thought to be split in the light, the carbon being used in the synthesis of organic matter and the oxygen being released into the atmosphere. This theory appeared to account adequately for plant photosynthesis, but a difficulty was presented by the observed behaviour of photosynthetic bacteria, discovered by Theodor Engelmann in the early 1880s s. Engelmann found that the purple bacteria (the first of which he called Bacterium *This interpretation of the ~sO-isotopestudies has since come into serious question (see Ref. 6). Otto Warburg [x,inted out that there is a continuous exchange of oxygen between CO2 and H20 by the formation and dissociationof H2COa(Ref. 7). If there is a large excess of H20 in these equilibration reactions, any oxygenisotopereleased in photosynthesisis likely to be derived from H20 regardlessof the nature of the photolyte.Warburgrevivedthe notionthat CO2 is reduced by the release of 02. Although few would now accept the details of Warburg~stheory, his criticisms of the interpretation of the ~sO-isotopestudies and his workon the catalytic role of CO2in chloroplast electron transport have led to further insight into the mechanismsof photosynthesis.