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Transcript
Chapter 13 C 4 Photosynthesis: Discovery, Resolution, Recognition, and Significance M.D. Hatch CSIRO Plant Industry, PO Box 1600, Canberra, ACT, 2601, Australia c.R. Slack Plant Physiology Division, Horticulture and Food Research Institute, Private Bag 11030, Palmerston North, New Zealand ABSTRACT 1his chapter provides an account of the events preceding and contributing to the discovery and the general acceptance of the biochemical option for photosynthesis now known as the C, pathway. We also trace the early stages of the elucidation of the mechanism of this process and its physiological significance. Relationships between plants using alternative biochemical processes for photosynthesis and such aspects as taxonomy, leaf anatomy, physiology and plant productivity, are briefly considered. Flow-on effects to other fields of the recognition of this alternative C, mechanism are identified. Background and Clues in Retrospect By the early 1960s, Calvin and his colleagues, Benson and Bassham, had resolved the essential details of the photosynthetic CO2 assimilation process known as the Calvin cycle or, more commonly now, the photosynthetic carbon reduction cycle (PCR cycle) or the C, pathway (see Andrew Benson, this volume). While most of the studies on the mechanism of this process were conducted with the alga Chiarella, there was reasonable evidence that a similar or identical mechanism accounted fm; CO2 assimilation by a number of higher plants. Based on the precedent of the universality of other biochemical processes it was, with good reason, generally assumed that this PCR cycle would account for photosynthetic carbon assimilation in all autotrophic species. In this chapter we will outline the events leading to the discovery of the existence of the biochemical variant of this PCR cycle, known as the C4 175 'j" :; I .• '. ::. I,' , Ii; :i~ j~ 176 ~I 1! M. D. Hatch and C. R. Slack pathway. We will follow this story through to the early stages of the resolution of the mechanism of this process, the recognition of its physiological significance, and to the point where the existence of this option for CO2 assimilation was generally accepted by the .wider plant research community. With the wisdom of hindsight, one can speculate that a very observant and insightful reader of the plant biology literature of the early 1960s might very well have concluded that a novel biochemical process for CO2 assimilation could be operating in some grasses of tropical origin, including maize (Zea mays) and sorghum. These species, all belonging to ,the grass sub-family Panicoideae, shared the unique anatomical, ultrastructural and physiological features listed in Table 1. Haberlandt (1904) was the first to recognise the unusual type of leaf anatomy featuring two distinct types of photosynthetic cells in certain groups of grasses. An example of this anatomy is shown in Fig. 1. He termed this specialised type of anatomy, with a layer of mesophyll cells outside a sheath of chloroplast-containing ceUs surrounding vascular elements, Kranz anatomy. His suggestion that these cells may have different functions was further supported by the these observations of Rhoades and Carvalho (1944) who particularly noted that starch accumulated only in the inner bundle sheath cells. They speculated, perhaps not quite correctly, that Table 1. Unique features shared by some tropicargrasses now known to be C, Date Feature Reference 1904 Specialised Kranz anatomy Haberiandt, 1904 1944 Bifunctional chloroplasts/cells Rhoades and Carvalho, 1944 1955 Dimorphic chloroplasts Hodge et al., 1955 1927 High water-use efficiency Shantz and Piemeisel, 1927 1962 Low CO2 compensation point Meidner, 1962; Moss, 1962 1963 High growth rates Loomis and Williams, 1963 1963 Leaf photosynthesis High maximum rates and light saturation Hesketh and Moss, 1963; Hesketh, 1963 High temperature optima .. EI-Sharkaway andHesketh, 1~64 C4 Photosynthesis 177 carbon was assimilated in the mesophyll cells but stored in the bundle sheath cells. Later, with the newly developed technique of electron microscopy, Hodge et al. (1955) demonstrated that the bundle sheath chloroplasts of maize had unusual ultrastructural features, with single unappressed thylakoid membranes and an almost total absence of grana. This turned out to be a feature of all NADP-ME-type C( grasses. Earlier, Shantz and Piemeisel (1927) reported a study of the efficiency of water use for carbon assimilation for a wide range of grasses and other plant species. For the grasses we now know as C(' the range of values for water lost per unit of dry matter fixed (g water per g dry matter produced) was 260 to 340 compared with 540 to 977 for the grasses we now know to be C3 • There followed a series of reports of unusual physiological features in this same sub-group of grasses, all rela:ting to photosynthetic function. For instance, there was evidence accumulating that these species had an unusually high potential for rapid growth (LOOmiS and Williams, 1963). They also showed very low CO2 compensation points (Moss, 1962; Meidner, 1962), close to zero cq compared with more than 50 ppm CO2 Fig. 1. Specialised 'Kranz' anatomy typical of C, leaves showing mesophyll cells (M), bundle sheath cells (BS) and vaScular tissue (VI. 178 M. D. Hatch and C. R. Slack for other species. This was coupled with unusually high leaf photosynthesis rates, only saturating at near full sunlight (Hesketh, 1963; Hesketh and Moss, 1963) and high temperature optima for photosynthesis (EI-Sharkaway and Hesketh, 1964; Murata and lyama, 1963). Radiotracer Evidence for an Alternative Pathway The coincidence of these unusual anatomical and physiological features in this group of grasses did not tum out to be the clue leading to the recognition of the C4 process for photosyntheSiS. That story begins another way. In the 1954 Annual Report of the Hawaiian Sugar Planters Association Experiment Station (HSPAES) there appeared a brief unreferenced comment, based on labelling sugar cane leaves with radioactive carbon dioxide, that "It seems clear that in sugar cane phosphoglyceric acid (PGA) is not the major carrier of newly fixed carbon that it is found to be in some algae." More details of this work appeared in an abstract of a report to the 1956/57 Annual Meeting of the Hawaiian Academy of Science presented by H.P. Kortschak, C.E. Hartt and G.O. Burr. In that report, one of three early labelled products was identified as phosphomalic acid. Later the same authors (Kortschak et al., 1965) said this compound was actually PGA. By 1960, further brief reports in the Annual Reports of the HSPAES identified two of the major early-labelled products in sugar cane leaves as malate and aspartate with PGA being the third. Time-course data suggested that malate was the first product of CO2 assimilation and that PGA and sugars were formed later. The same data was very briefly mentioned by George Burr in a paper on the use of radioisotopes by Hawaiian sugar plantations at a Pacific Science Congress symposium. The proceedings were published in the International Journal of Applied Radiation and Isotopes (Burr, 1962). At about this time in 1960 a young Russian scientist, Yuri Karpilov, found similar labelling patterns when maize leaves were exposed to HC02 • in the light. The results were reported in the Proceedings of the Kazan Agricultural Institute (Karpilov, 1960). These results clearly showed malate and aspartate as the major early-labelled products in illuminated maize leaves, with PGA and sugar phosphates being more rapidly labelled after longer periods. The conservative conclusion was, according to the translation, that the radio labelling in maize leaves "was not characteristic of other species of plants". In 1963, Karpilov co-authored a paper (Tarchevskii and Karpilov, 1963) examining the effect of different killing procedures on the radiolabelled products of photosynthesis. They apparently concluded that aspartate and PCA at least, are formed by the degradation of other labelled compounds when leaves are killed in boiling ethanoL The implication for his earlier work was not clear. Karpilov published other papers on nitrogen and phosphorous metabolism in leaves but it was not until 1969 that he published further on the mechanism of photosynthesis in maize and related grasses (Karpilov, 1969). Needless to say, the Hawaiian and Russian workers remained unaware of each other's work for several years to come and we remained unaware of the Russian work until about 1969. In the early 1960s we were working in the laboratory of the Colonial Sugar Refining Company in Brisbane, Australia, on aspects of carbohydrate metabolism in sugar cane. Researchers in this laboratory were in regular contact with staff of the HSPA Experiment Station about a range of matters relating to sugar cane research. As a result we were aware of the work reported in the Annual Reports on the labelling of compounds from photoassimilated 14C02, and had often discussed the possible implications of this observation. Oddly, it was not until 1965 that this Hawaiian work was published in a form accessible to the research community (Kortschak et al., 1965). This first detailed report showed kinetics of incorporation of 14cq consistent with malate and aspartate being the first major products of cq assimilation, with PCA and sugar phosphates being rapidly labelled onlyafter a lag of several seconds. Sucrose and an insoluble component (probably starch) were labelled after about 60s. They concluded that "carbon assimilation proceeds by a path qualitatively different from many other plants". In a following brief report, Kortschak and Hartt (1966) noted that this unusual labelling pattern in sugar cane was similar when light, CO2 concentration and leaf age were varied. Later in 1965, actually over a glass of beer at a scientific meeting in Hobart, we decided we should repeat and extend these observations on sugar cane to resolve the question of their precise meaning and significance. In designing these experiments we paid particular attention to conducting the "C02 labelling under steady-state conditions for photosynthesis, and with high light and physiological concentrations of CO2 , Our results for the kinetics of time-course labelling from 14C02 (Hatch and Slack, 1966) were similar to those reported by Kortschak et al., (1965) for sugar cane. As it turned out, they were also similar to those reported . by Karpilov (1960) for maize but, as we already said, we were not aware of this work until about 1969. We extended these studies in several ways. Most critical were pulse-chase experiments where leaves are labelled with HC02 for a period and then transferred to normal air containing unlabelled CO2, The movement of previously fixed radioactivity between compounds 180 t: I ,i: M. D. Hatch and C. R. Slack is then followed during the chase period. This kind of experiment, conducted under steady-state conditions, demonstrated the rapid movement of carbon from the dicarboxylic acid malate to PGA and then to hexose phosphates and finally the end-products sucrose and starch. Such data unambiguously demonstrated for the first time that the dicarboxylic acids were intermediates in the formation of PGA and its products; this essentially ruled out the possibility that labelling of the C4 acids may have been due to reversible exchange reactions not connected with the net assimilation process (Hatch and Slack, 1966). Using special killing procedures we were able to identify oxaloacetate as a minor but rapidly labelled product; its kinetics of labelling were at least as rapid as the other C4 acids. Furthermore, by isolating and degrading labelled intermediates we showed that (i) malate and aspartate were initially labelled exclusively in the C-4 carboxyl carbon, (ii) that this carbon gives rise to the C-1 carboxyl of PGA, and (iii) that the labelling pattern in hexoses was consistent with their formation from PGA via the conventional PCR cycle (Hatch and Slack, 1966). From this data we developed a simple working model to explain photosynthesis in sugar cane (Hatch and Slack, 1966) and this is reproduced in Fig. 2. It proposed that the initial assimilation of CO2 occurs by carboxylation of a 3-carbon compound, probably PEP or pyruvate, giving rise to a C. dicarboxylic acid with the C-4 carboxyl derived from COr Which of the three acids, malate, oxaloacetate or aspartate was the primary product of the carboxylation reaction could not be said with certainty but, in any case, these acids were apparently rapidly interconverted. Regarding the transfer of the C-4 carboxyl of C4 acids to the C-1 of PGA we now know that we were wrong in favouring the idea that this proceeded via a trans carboxylation reaction. We considered the pOSSibility that this occurred by decarboxylation of the C4 adds and refixation of the released CO2 but thought this to be unlikely on the grounds that the transfer would be too inefficient due to diffusive, loss of CO2 (Hatch and Slack, 1966). As will be discussed in more detail below, our later erroneous conclusion that these tropical grasses contained very low Rubisco activities (Slack and Hatch, 1967) provided apparent support for this idea. Of course, it was some years before the full Significance of the Kranz anatomy and CO2-tight bundle sheath cells became evident. The other critical feature of the scheme in Fig. 1 was the suggestion that a cyclic process operates to initially fix CO2, with the primary acceptor being regenerated from the 3-carbon compound remaining after the transfer of the C-4 carboxyl of C4 adds to PGA. 0 4 Photosynthesis 181 Soon after, we named this process the C. dicarboxylic acid pathway of photosynthesis (Hatch and Slack, 1968) and this became abbreviated to C4 pathway or C. photosynthesis. Later, the term Ca photosynthesis was used as an alternative to PCR cycle or Calvin cycle, and species using these pathways became known as C, and Ca plants, respectively. The history of the development of these abbreviated terms is somewhat obscure. At an international meeting held in Canberra towards the end of 1970 these terms were used by most participants (see Hatch et al., 1971). However, we could find only one paper published prior to that meeting using similar terms; that was a paper by Osmond et al. (1969) who referred to 'Ca-type' and 'C, type' plants and photosynthesis. Sucrose i i Hexose phosphates diphosphate 1) carboxyl acceptor 3C 3-Phospho- ) glyc~rate . : ~bulose 2~ . I IC· I t Aspartate 'hO(:'::I1~~a,. } 3C 1 2C+C*02 1 IC 1l 4C* ;{ --1- 3C I Oxaloacetate 2C 1 IC H Malate Scheme 1. Proposed pathway for photosynthetic fixation of carbon dioxide in sugar-cane leaves. The broken arrow indicates a minor pathway. Fig.2. Working model for C. photosynthesis proposed in 1966 on the basis of the kinetics and intramolecular location of "CO, incorporated into intermediates and products by sugar cane leaves (Hatch and Slack, 1966). Resolution of the Mechanism and Function During 1967 we were joined by two PhD students from the University of Queensland, Hilary Warren and John Andrews. They contributed in many 182 M. D. Hatch and C. R. Slack important ways to our investigations of the C( process during the following three years. In our second paper (Hatch et al., 1967) we confirmed that C. acids remained the dominant early-labelled products when leaf age, CO, concentration and light were varied. In fact, when light was reduced to about 1/10 of full sunlight (200 J..l.mol quanta m"s'I), 1(C labelling was not detectable in compounds other than malate and aspartate after a 3s exposure to 14C02. More importantly, that paper reported similarly high proportions of fixed HC in malate and aspartate (with oxaloacetate) in several grass species from different tribes, indicating that the process was common in the Gramineae at least. Of course, normal C3-type labelling patterns were seen for many other species from Grarnineae and other families. Although it appeared unlikely that this process might be unique to sugar cane, this was still a very exciting result for us. Significantly, a sedge (Cyperus, family Cyperaceae) also showed classical C. acid labelling. In the same year, Osmond (1967) reported similar labelling in the dicotyledenous species, Atriplex spongwsa (salt bush), and later we showed the C. dicarboxylic acid labelling pattern in several other dicotyledonous species from different plant families (Johnson and Hatch, 1968). Clearly, these proposals outlined in the scheme in Fig. 1 had to be consolidated with further evidence, especially with regard to the enzymes involved. This scheme provided a basis for a cycle of predictions, discoveries of enzymes and more predictions. A prediction from the radiotracer studies was that either PEP or pyruvate was the primary CO, acceptor. A search for appropriate enzymes revealed two likely contenders, PEP carboxylase and NADP-maIic enzyme (which can catalyze the reductive carboxylation of pyuvate to malate) both of which were at least twenty times more active in the leaves of C. grasses compared with C3 species (Slack and Hatch, 1967). These enzymes catalyse the following reactions: PEP carboxylase:- PEP + CO2 4 Oxaloacetate +Pi NADP malic enzyme:- Malate + NADP ~ Pyruvate + CO, + NADPH We favoured PEP carboxylase as the primary carboxylating enzyme; it was some time before the decarboxylating function of NADP-malic enzyme in these C. species was fully recognised (Berry et al., 1970; Hatch, 1971a; Hatch and Slack, 1970b). With PEP as the likely CO2 acceptor, it seemed that C( leaves should be capable of converting pyruvate to PEP. The only known plant enzyme capable of this conversion was pyruvate C4 Photosynthesis 183 kinase but its activity in the reverse direction was far too low. However, Cooper and Kornberg (1965) had just described a bacterial enzyme which converted pyruvate to PEP but generated AMP and Pi as the other products instead of ADP: Pyruvate + ATP ~ PEP + AMP + Pi We searched for and fInally detected a reaction with apparently the same stoichiometry in leaves of tropical grasses (Hatch and Slack, 1967). This was an exciting discovery. Furthermore, the fact that this activity was not detectable in the leaves of C. plants provided additional support for the idea that PEP was the primary CO2 acceptor. Of course, following studies (Hatch and Slack, 1968) were to reveal that this PEP synthesising reaction was, in fact, much more complicated with Pi as a co-substrate and PPi a product: Pyruvate + ATP + Pi ~ PEP + AMP + PPi This reaction was unique in phosphorylating two substrates, pyruvate and Pi, from the ~ and 'Y phosphates of ATP, respectively. Accordingly, the enzyme was named pyruvate, Pi dikinase. We initially failed to detect the requirement for Pi because it was being added accidentally to the reaction with the coupling enzyme and the production of PPi was not observed because of contaminating pyrophosphatase activity. With the identification of AMP and PPi as the other products of this reaction it was apparent that they should be rapidly recycled. A search for the most obvious enzymes to achieve this, adenylate kinase and pyrophosphatase, revealed that activities in C( leaves were some 40 to 60 times those in C.leaves (Hatch et al., 1969). In mid-1967 the momentum of our collaborative research was temporarily interrupted when one of us (MDH) accepted a new appointment with teaching and administrative duties in the Botany Department at the local University and CRS began unrewarding experiments in search of the trans carboxylation reaction which we had proposed from our initial studies (see Fig. 2). A more productive direction of research was taken, some months later, following an informal meeting with colleagues at CSIRO in Canberra. Here, the possibility of metabolic differences between the morphologically distinct mesophyll and bundle sheath chloroplasts, associated with the Kranz anatomy of tropical grasses, was discussed. As a result, we decided to investigate the distribution of enzymes between the two chloroplast types using non-aqueous density 184 M. D. Hatch and C. R. Slack fractionation of freeze-dried leaf tissue. These studies showed that pyruvate, Pi dikinase was exclusively localised in mesophyll chloroplasts whereas Rubisco and phosphoribulokinase were restricted to the bundle sheath cell chloroplasts. Unfortunately, difficulties were encountered in publishing these rather novel fmdings, with the implication that the two chloroplast types have differing metabolic functions and with Rubisco totally absent from the mesophyll chloroplasts. As a result, the paper describing these initial findings (Slack, 1969) only appeared at about the same time as our far more comprehensive account of chloroplast enzyme complements (Slack et al., 1969). A fortunate biproduct of this study was the finding that pyruvate, Pi dikinase was barely detectable in leaves taken directly from darkness and this observation led to the discovery of the dark-inactivation and light activation of the enzyme in vivo (Slack, 1968). After his brief sojourn in University life, MDH returned to the Brisbane laboratory. We renewed our collaboration by studying this light-dark regulation of pyruvate, Pi dikinase and showing that Pi was required for the activation of the enzyme from darkened leaves and that dark inactivation was probably due to a reaction with ADP (Hatch and Slack, 1969a). The full complexity of these processes was only resolved many years later (see Burnel1 and Hatch, 1985). Assuming that oxaloacetate was the first product of Co. fixation there was also the critical question of how this was reduced to give the large pools of malate seen when 14CO, was fixed by leaves. The leaves of these tropical grasses like maize and sugar cane contained high levels of NAD specific malate dehydrogenase but its activity was no greater than in leaves of several C3 plants (Slack and Hatch, 1967). Furthermore, there was the question of how such high levels of NADH production might be sustained. Consequently, we looked for a reaction involving NADPH, since it could be generated directly by light-dependent reactions in chloroplasts. As a result, we showed that leaves of tropical grasses contained a novel NADP specific malate dehydrogenase (Hatch and Slack, 1969b) catalysing the following reaction: Oxaloacetate + NADPH -+ Malate + NADP Early difficulties in measuring consistent levels of this enzyme were resolved when it was realised that it resembled pyruvate, Pi dikinase in imdergoing dark~light mediated inactivation and activation in leaves (Johnson and Hatch, 1970). Of course, we originally thought that this activity may be unique to C. plants but this did not tum out to be the case. C4 Photosynthesis 185 However! its activity in the NADP-ME-type C. species in particular, was about ten times that in leaves of plants or indeed most other plants (Hatch et al., 1975). During the period between 1966 and mid-1969 we worked largely in isolation .from the rest of the world and were unaware of the gathering research effort on C. photosynthesis going on elsewhere. In fact, during this time, our papers were the only ones published on the biochemistry of C. photosynthesis. This situation could not last and the change for us occurred when we attended the International Botanical Congress in Seattle in the summer of 1969. Here we met others who were working on this topic and learned that the questions of the role of Rubisco in C. plants, and also the inter- and intracellular location of other photosynthetic enzymes, were on the minds of the increasing number of players in the C. photosynthesis field. Important to these questions was the recognition by then that the 'Kranz' anatomy (see Fig. 1), described so many years before (Haberlandt, 1904), was apparently a specific feature of all the C. species then identified. As already mentioned, our earlier analysis of Rubisco in extracts of C~ leaves had indicated low activities compared with Cl leaves (Slack and Hatch, 1967). One of those we met at the Seattle Congress was Olle Bjorkman who told us that they had shown that Rubisco activities in leaf extracts of C. plants were similar to the activities recorded in C3 leaves and that this activity was largely confined to the bundle sheath cells. These studies, then in press (Bjorkman and Gauhl, 1969), also explained the discrepancy between the results by showing that few bundle sheath cells were broken during the Waring blending extraction procedure that we used in earlier studies. In fact, this blending procedure provided a very useful method for preparing intact bundle sheath cells as strands surrounding vascular tissue, and free of mesophyll cells. This method was refmed and first used for metabolic studies by Edwards et al. (1970) and Edwards and Black (1971). Such cell preparations turned out to be critical in a wide range of studies on bundle sheath cell metabolism (see Hatch, 1987). Also in press at that time was our work (Slack et al., 1969) showing that, in maize leaves, pyruvate, Pi dikinase and NADP-specific malate dehydrogenase were located exclUSively, and adenylate kinase and pyrophosphatase predominantly, in mesophyll chloroplasts. By contrast, Rtibisco, most other PCR cycle enzymes and NADP malic enzymes were located in bundl.e sheath chloroplasts. This data, taken together with the data of Bjorkman and Gauhl (1969) and also Berry et at., 1970), demonstrated how the chloroplasts in the two types of cells must act cooperatively in the overall C. photosynthetic process. It followed that the 186 M. D. Hatch and C. R. Slack movement of carbon from C, acids to 3-PGA almost certainly occurred exclusively in the bundle sheath chloroplasts by the decarboxylation of malate by NADP-malic enzyme followed by the refixation of the released CO2 via Rubisco. Thus, the need to consider a transcarboxylation reaction to account for this transfer was largely eliminated. Apparently, the main photosynthetic function of the mesophyll cells was to fix CO2 via PEP carboxylase and to generate PEP and reduce oxaloacetate in the chloroplast via pyruvate, Pi dikinase and NADP-specific malate dehydrogenase, respectively. A major implication of this was that malate must move from mesophyll chloroplasts to bundle sheath chloroplasts with a stochiometric amount of pyruvate moving in the reverse direction. During 1969, we wrote two reviews on aspects of the emerging C, storY and related matters (Hatch and Slack, 1970a, 1970b). The next question was what could the purpose be of fixing CO2 into C, acids only to regenerate CO2 by decarboxylation and refix it again via Rubisco? Suggestions at an International meeting held in Canberra in 1970 that this may be part of a mechanism to concentrate CO2 in bundle sheath cells (Bjorkman, 1971; Hatch, 1971a) were supported by the subsequent evidence that large pools of CO2 /HCOg develop in C. leaves in the light (Hatch, 1971b). The precise implications of such high bundle sheath CO2 concentations for Rubisco oxygenase activity and photorespiration in C, leaves was to emerge slowly over the next several years (Chollet and Ogren, 1975; Hatch and Osmond, 1976). By 1970 it was possible to formulate a detailed scheme describing the mechanism of carbon assimilation in those C. species like maize and sugar cane, characterised by having a high level of NADP malic enzymes in bundle sheath cells (Andrew et al., 1971; Hatch, 1971a). The scheme, presented at an international meeting on photosynthesis held in Canberra in 1970 (see Proceedings, Hatch et al., 1971), is reproduced in Fig. 3. By the time of this meeting, the existence of thisC. option for CO2 assimilation had gained wide acceptance. It could be then claimed that the process and its basic mechanism had not only been discovered but recognised and accepted by the wider plant research community. Briefly, this scheme proposed that CO2 is initially fixed in mesophyll cells via PEP carboxylase and that the oxaloacetate so formed is converted to larger pools of malate or aspartate via NADP malate dehydrogenase or aspartate aminotransferase. At least in NADP-ME-type species, malate then moves to bundle sheath cell chloroplasts where it is decarboxylated via NADP malic enzyme. While the released CO2 is assimilated via Rubisco and the PCR cycle, pyruvate is returned to mesophyll cells to serve as a precursor for the regeneration of the PEP. The latter reaction is 04 Photosynthesis 187 catalysed by the remarkable pyruvate, Pi dikinase in the mesophyll chloroplasts. As the scheme in Fig. 3 infers, the C. acid cycle is a metabolic appendage to the PCR cycle, acting essentially as a CO2 pump. As it later emerged, this provided Rubisco with the luxury of operating with minimal oxygenase activity. This scheme for NADP-ME-type C. photosynthesis remains substantially unaltered (see Hatch, 1987). The exclusive location of the C. acid generating reactions in mesophyll cells, and of Rubisco and the PCR cycle in bundle sheath cells, turned out to be common to all C. species. However, as briefly mentioned below, three different subgroups of C. plants emerged, distinguished by different mechanisms for the decarboxylation of C. acids in bundle sheath cells . "( >- - . :r ~ UJ :r (J) UJ ...J a z ::::> III I :2 /FRUCTOSE 6-P _ _ SUCROSE+STARCH TRIOSE t PHOSPHAT~PGA~ NADP NADPH' M~f;\ V MALAfE P'NTOS.?HOSPHAT'S Ru 5-P ~ Ii/ ( ~ 1==1 ALA\' ASPA~' PYRUVATE-rALANINE ASPARTATE p~~~e::7:A::.t:;~-N:2~ ""PHOSPHb.PYRUVATE ADP • OXALOACETATE Fig. 3. A scheme presented in 1970 to describe the path of CO, assimilation in C, species with high NADP malic enzyme (left side) and other C, species (right). From Hatch (1971a). Subsequent Developments on the Mechanism and Function If this NADP-ME-type C. mechanism operating in sugar cane and maize had turned out to be common to all C. species, then it would have only 188 M. D. Hatch and C. R. Slack remained to determine the properties of the enzymes involved and the regulation of the process. In fact, C4 photosynthesis was a lot more complicated. TIuee different groups of C4 plants emerged, defined by the operation of either NADP-malic enzyme, NAD-malic enzyme, (Hatch and Kagawa, 1974; Hatch et al., 1975) or PEP carboxykinase (Edwards et al., 1971; Gutierrez et al., 1974) for decarboxylating C4 acids (Table 2). This was a most unexpected outcome, made even more surprising by the fact that each of these reactions was located in a different cell compartment and was accompanied by its own suite of associated enzymes (Table 2). These mechanisms took years to resolve and progress towards their resolution can be followed in a series of reviews and books (Black, 1973; Hatch and Osmond, 1976; Edwards and Huber, 1981; Edwards and Walker, 1983; Hatch, 1987) and a number of subsequent papers. In fact, aspects of these areas remain the topic of current research. Table 2. The location and activity of enzymes in the subgroups of C, species defined by the operative C, acid decarboxylase. 'High' and 'Low' refer to enzyme activities relative to other groups of C, plants or to C. plants. For details see Hatch (1987). Abbreviations: Mal, malate; Pyr, pyruvate, OM oxruoacetate. NADP-ME- TYPE PCK-TYPE NAD-ME-TYPE Reaction MaI-4Pyr+COa OAA-4PEP+COa Mal-4Pyr+COz Location Chloroplast Cytosol Mitochondria NADP malic enzyme High Low Low PEP carboxykinase Low High Low NAD malic enzyme Low Low High NADP-MDH* High Low Low Aminotransferases Low High High PEP carboxylase High High High PPDK* High High High Decarboxylase Other enzymes • NADP-MDH, NADP malate dehydrogenase; PPDK, pyruvate, Pi dikinase. C4 Photosynthesis 189 The precise function and advantages of the C. option for photosynthesis were also more clearly focused and defined in the following years. As the significance of concentrating CO2 in relation to Rubisco oxygenase activity emerged, it appeared that there would be little photorespiration in C( leaves under normal conditions (Chollet and Ogren, 1975; Hatch and Osmond, 1976; Edwards and Walker, 1983; Hatch, 1987). More recently, conditions under which this might not apply have been defined (Dai et al., 1993, 1995). The implications of reduced photorespiration for quantum requirements (Farquhar, 1983; Furbank et al., 1990) and temperature responses (see Hatch, 1992) of C( photosynthesis have been discussed. In related studies, estimates have been made of the CO2 concentration in bundle sheath cells in the light (Jenkins et al., 1989; Dai et al., 1995), the permeability of the mesophyll-bundle sheath cell interface to CO2 (see Jenkins et al., 1989) and the leakage of CO2 from bundle sheath cells during photosynthesis (Henderson et al., 1992; Hatch et aI.,1995). Relation to Physiological Features and Performance of Plants We now know that the anatomical, physiological and performance features listed in Table 1 are causatively linked with the C. photosynthetic process and are invariably associated with this process. As we said at the beginning of this chapter, recognition that these various features were invariably linked might have led a curious and insightful researcher to investigate whether the CO2 assimilation process was different in this group of plants. In fact, by the time some of these links were being recognised (EI-Sharkway and Hesketh, 1965) the first evidence for a new pathway in sugar cane and related tropical grasses was already appearing (Kortschak et al., 1965; Hatch and Slack, 1966). The fact that species showing this set of anatomical and physiological features invariably fixed CO2 by the new C. pathway was recognised by several groups of researchers by 1968 (Hatch et al., 1967; Downes and Hesketh, 1968; Downton and Tregunna, 1968; Johnson and Hatch, 1968; Laetsch, 1968). In an important paper, Black et aI., (1969) drew together the available information correlating the options for photosynthetic CO2 assimilation with these other features. They discussed the link between the more efficient C. photosyntheSis, competitiveness, and productivity, and pointed out that a high proportion of the most common weeds are C. plants. It was already clear by 1970 that the specialised Kranz leaf anatomy was critical for the operation of C. photosynthesis. Later, the reasons why the operation of C. photosyntheSiS accounted for the various unique 190 M, D, Hate/; and G, R. Slack physiological features listed in Table 1 were to emerge (Edwards and Walker, 1983; Hatch, 1987; Hatch, 1992). In particular, it should be emphasised that the C. photosynthetic option provides plants with the potential for record productivity, substantially exceeding that of any C3 plant (Hatch, 1992). What has confused this issue is that this potential will only be realised under appropriate environmental conditions. Furthermore, it will not be evident in all C. species, many of which evolved not to capitalise on the capacity for rapid growth but for survival where water is limiting. ' Taxonomy and Evolution of C. Species By 1970, a large number of grass species from the Pani~o,ideae and Eragrostoideae subfamilies were recognised to be C. plantsidong with some species from another monocotyledonous family Cyperaceae (Downton, 1971). There was also a rapidly increasing number of dicotyledonous species being, added to this list from eight different families: Azioceae, Amaranthaceae, Chenopodiaceae, Compositeae, Euphorbiaceae, Nyctaginaceae, Portulaceae and Zygophyllaceae. At that time Evans (1971) made the important point that C. plants occur only in the advanced, more recently evolved Orders. It was also dear that they were diverse Orders and that the process must have evolved separately on many occasions. It appears likely that these species have evolved during the last 50 million years or so, in response to the pressures of a dramatic decline in atmospheric CO2 concentration (Ehleringer et al., 1991). Many more C. species have now been identified and the number of contributing dicotyledonous families has increased to fourteen (Edwards and Walker, 1983). It has been estimated that there are at least 7500 C. species, still only about 3% of the total number of Angiosperm species. Another important development has been the recognition of the existence of a small number of species with characteristics intermediate between C3 and C. (Edwards and Ku, 1987; Rawsthome, 1992). The origin and characteristics of these species dearly has great significance with regard to the evolution of C. photosynthesis. Hattersley and Watson (1992) have prOVided a very detailed analysis of the relationships between the photosynthetic biochemistry, physiology, anatomy, taxonomy and evolution of the grass species using different mechanisms for CO2 assimilation. 04 Photosynthesis 191 Implications for Other Fields and Further Discoveries As the C. story unfolded it has left a trail of important discoveries in their own right and developments in related fields. For instance, at the biochemical level, two new enzymes, pyruvate, Pi dikinase (Hatch and Slack, 1968) and NADP-malate dehydrogenase (Hatch and Slack, 1969b) were discovered in the course of resolving the mechanism. Furthermore, the double phosphorylation mechanism of the reaction catalysed by pyruvate, Pi dikinase was almost unique in the field of biochemistry as were aspects of the mechanism by which this enzyme was dark/light regulated (Burnell and Hatch, 1985). This regulation involved an unprecedented phosphorylation of the enzyme by ADP leading to inactivation and reactivation by the phosphorolytic removal of this phosphate group to yield pyrophosphate, rather than the usual hydrolysis. In addition, these fwo mechanistically different reactions were catalysed by the same enzyme. It is also fair to say that the resolution of the biochemical mechanisms for CO2 assimilation via Crassulacean Acid Metabolism was very much aided by prior knowledge of the enzymes and pathways of C. photosynthesis (Osmond and Holtum, 1981; Ting, 1985). There are remarkable similarities between the biochemical pathways of the night time fixation of CO2 via the Crassulacean Acid Metabolism mechanism and the C. pathway. As has been noted before, in the C 4 pathway fixation of CO2 into C. acids, the subsequent release and refixation of this CO2 in bundle sheath cells is separated in space whereas, in the former process, the separation is in time. This was recognised quite early when Laetsch (1971) referred to C. plants as "CAM mit Kranz". Investigations of the mechanism of C. photosynthesis have also thrown further light on the role of plasmodesmata in intercellular metabolite transport and the permeability of cells walls to CO2 , Laetsch was first to note the profusion of plasmodesmata connecting mesophyll and bundle sheath cells (Laetsch and Price, 1968; Laetsch, 1971) and later studies provided direct evidence for the high permeability of this cell interface to metabolites (see Hatch, 1987, 1992). Cell. walls are normally freely permeable to CO2 but the flux of CO2 across the outer surface of bundle sheath cells is restricted; at least in some cases this is linked with the presence of a suberin lamellae (see Hatch, 1987; Jenkins et al., 1989). The existence of C. plants, with their range of unusual physiological and performance features, has provided a strong stimulus to the 192 M. D. Hatch and C. R. Slack consideration and understanding of the basis of plant pe;rforman;;e. For instance, information on C, plants has been an important component in understanding the basis of the responses or relationships of photosynthesis to varying temperature, quantum yield, water-use, photorespiration and, indeed, varying productivity (see Beadle and Long, 1985; Hatch, 1987, 1992; Nobel,1991). Recognition of the C, process and its biochemical sub-g:roups has been of special interest with respect to taxonomy, especially of the g:rasses. By and large, there has been a clear cong:ruency of C 3 and C, plants, and also the C, subgroups, with existing taxonomic g:roupings. However, where anomalies have occurred, they have in some cases led to reconsideration of existing taxonomic classifications (Brown, 1977; Webster, 1987). Finally, some unexpected spin-offs of the recognition of C, photosynthesis and the follow-on studies. These concern applications of the finding that C, plants discriminate less against the heavier isotopes of carbon during COl assimilation compared with C3 plants (Tregunna et aI., 1970; Smith, 1972). As a result, C3 and C, plants have different ratios of 12C to 13C or I'C in their organic matter. This knowledge has been used in assessing the COl composition of prehistoric atmospheres (Marino et al., 1992) and testing 'for adulteration of foods and alcoholic beverages (Doner and Bills, 1981; Anon, 1993). For instance, wine, beer and whiskeys are derived from C3 plant products and should contain ethanol with a C3-like ratio of 13C to 12c. If the ratios are higher then this would indicate that a C, derived sugar or com syrup has been added. It is interesting to note that this difference also has implications for the use, in archaeology and other fields, of carbon isotope dating, based on the prevailing content of the unstable isotope, Uc. The original content of the unstable isotope utI would be g:reater in C,-derived organic matter compared with C3 leading to an underestimation of age. This difference can be corrected, and therefore the estimated age of material adjusted, by also estimating the ratio of the stable isotope 13C to nC. References Andrew, T.]., Johnson, H.S., Slack, C.R. and Hatch, M.D. (1971) Malic enzyme and <'lminotransferases in relation to 3-phosphoglyc~rate formation in plants with the C, dicarboxylic add pathway of photosynthesis. Phytochemistry 10: 2005-2013. Anonymous (1993) Analytical News, Autumn issue. Beadle, C.L. and Long, S.P. (1985) Photosynthesis is it limiting biomass production? Biomass B: 119-168. C4 Pho tosynthesis 193 Berry, 1.A., Downton, W.J.S. and Tregunna, E.B. (1970) The photosynthetic carbon metabolism of Zea mays and Gcnnphrena globosa: the location of the CO, fixation and carboxyl transfer reactions. Can. J. Bot. 48: 777-786. Bjorkman, 0. (1971) Comparative photosynthetic CO, exchange in higher plants. In Photosynthesis and photorespiratian, M.D. Hatch, C.B. Osmond and R.O. Slatyer, eds., pp. 18 32 Wiley-Interscience, New York. Bjorkman, 0. and Cauhl, E. (1969) Carboxydismutase activity in plants with and without f3 carboxylation photosynthesis. Planta 88: 197-203. Black, C.C. (1973) Photosynthetic carbon fixation in relation to net CO, uptake. Annu. Rev. Plant Physiol. 24: 253-286. Black, c.c., Chen, T.M. and Brown, RH. (1969) Biochemical basis for plant competition. Weed Sci. 17: 338-344. Brown, W.V. (1977) The Kranz syndrome and its sub-types in grass systematics Memoirs Torrey Bot. Club. 23: 1-97. Burnell, J.N. and Hatch, M.D. (1985) Light-dark modulation of leaf pyruvate, Pi dikinase. Trends in Biochemical Sciences. 10: 288-291. J. Applied Radiation Isotopes, 13: 365-374. Chollet, R and Ogren, W.L. (1975). Regulation of photorespiration in C, and C, species. Botan. Reviews 41: 137-179. Cooper, RA. and Kornberg, H.L. (1965) Net conversion of pyruvate to phosphoenol pyruvate in Escherichia coli. Biochim. Biophys. Acta 104: 618-621. Dai, Z., Ku, M.S.B. and Edwards, G.E. (1993) C, photosynthesis: the CO, concentrating mechanism and photorespiration. Plant Physiol. 103: 83-90. Dai, Z., Ku, M.S.B. and Edwards, C.E, (1995) C, photosynthesis: Effect of leaf development on the CO, concentrating mechanism and photorespiration in maize. Plant Physiol. 107: 815~ Burr, C.O. (1962) The use of radioisotopes by Hawaiian Sugar Plantations. Inst. 825.. Doner,'L.W. and Bills, D.O. (1981) Stable carbon isotope ratios in orange juice. Chern. 29: 803-804. J. Agric. Food . Downes, R.W. and Hesketh, J.D. (1968) Enhanced photosynthesis at low 0, concentrations. Differential response of temperature and tropical grasses. Pianta 78: 79-84. Downton, W.I.S. (1971) Check-list of C, species. In Photosynthesis and Photorespiration. M.D. Hatch, C.B. Osmond and RO. Slatyer, eds., pp 554-558. Wiley-Intersdence, New York. Downton, W.J.S. and Tregunna, E.B. (1968) Carbon dioxide compensation - its relation to photosynthetic carboxylation reactions, systematics of Crarnineae and leaf anatomy. Can. J. Bot. 46: 207-215. Edwards, C.E. and Black, c.c. (1971) Isolation of mesophyll and bundle sheath cells from Digitaria sanguinalis (L) Seop. leaves and a scanning microscope study. Plant Physiol. 47: 149-156. Edwards, C.E. and Huber, S.c. (1981) The C, pathway. In The Biochemistry of Piants. Vo!' 8, Photosynthesis. M.D. Hatch and N.K. Boardman, eds., pp. 237-281. Academic Press, New York. Edwards, C.B., Kanai, R and Black, c.c. (1971) Phosphoenolpyruvate carboxykinase in leaves of <:ertain plants which fix CO, by the C, dicarboxylk cycle of photosynthesis. Biochem. Biophys Res. Ccnnmun. 45: 278-285. Edwards, G.E. and Ku, M.S.B (1987) Biochemistry of C.-C, Intermediates. In The Biochemistry of Plants. Vol. 10, Photosynthesis. M.D. Hat<:h and N.K. Boardman, eds., pp. 275-325. Academic Press, New York. Edwards, G.B., Lee, 5.S., Chen, TM. and Black, c.c. (1970) Carboxylation reactions and photosynthesis of carbon compounds in isolated mesophyll and bundle sheatli cells of Digitaria sanguinalis (L) Seep. Biochem. Biophys. Res. Commun. 39: 389-395. 194 M. D. Hatch and C. R. Slack Edwards, G.B. and Walker, D.A. (1983) c.-C,: Mechanisms and Cellular and Environmental Regulation of Photosynthesis. Blackwell Scientific Publications, Oxford. Ehleringer, J., Sage, R.F., Flanagan, L.B. and Pearcy, R.W. (1991) Climate change and ,the evolution of C, photosynthesis. Trends in Ecol, Eval. 6: 95-99. El-Sharkaway, M.A. and Hesketh, J.D. (1964) Effect of temperature and water deficit on leaf photosynthetic rates of different species. Crop Sci. 4: 514-518. El-Sharkaway, M.A. and Hesketh, J.D. (1965) Photosynthesis among species in relation to characteristics of leaf anatomy and CO, diffusion resistance. Crop Sci. 5: 517-521. Evans, L.T. (1971) Evolutionary, adaptive and environmental aspects of the photosynthetic pathways. In Photosynthesis and Photorespiration. M.D. Hatch, C.B. Osmond and R.O. Slatyer, eds., pp. 130-136. Wiley Interscience, New York. Farquhar, G.D. (1983) On the nature of carbon isotope discrimination in C, species. Aust. J. Plant Physiol. 10: 205-226. Furbank, R.T., Jenkins, C.L.D. and Hatch, M.D. (1990) C, photosynthesis: Quantum requirement, C, acid overcyding and Q cycle involvement. Aust. J. Plant Physiol. 17: 1-7. Gutierrez, M., Gracen, V.E. and Edwards, G.E. (1974) Biochemical and cytological relationships in C, plants. P!anta 19: 279-300. Haberlandt, G. (1904) Physiologische Pflanzenanatomie. Wilhelm Engelman, Leipzig. Hatch, M.D. (1971a) Mechanism and function of C, photosynthesis. 1n Photosynthesis and Photorespiration. M.D. Hatch, C.B. Osmond and R.O. Slatyer, eds., pp.139-152 Wiley Interscience, New York. Hatch, M.D. (1971b) The C, pathway of photosynthesis. Evidence for an intermediate pool of CO, and the identity of the donor C, dicarboxylic acid. Biochem. J. 125: 425-432. Hatch, M.D. (1987) C, photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim. Biophys. Acta 895: 81~106. Hatch, M.D. (1992) The making of the C, pathway. 1n Research in Photosynthesis. N. Murata, ed., Vol. 3, pp. 747-756. KIuwer Academic Publishers, Netherlands. Hatch, M.D., Agostino, A. and Jenkins, C.L.D. (1995) Measurement of the leakage of CO, from bundle sheath celis of leaves during C, photosynthesis. Plant Physiol. 108:173-181. Hatch, M.D. and Kagawa, T. (1974) Activity, location and role of NAD malic enzyme in leaves with C, pathway photosynthesis. Aust. J. Plant Physiol. 1: 357-369. Hatch, M.D., Kagawa, T. and Craig, S. (1975) Subdivision of C, pathway species based on differing C4 acid decarboxylating systems and ultrastructural features. Aust. J. Plant Physicl. 2: 111-128. Hatch, M.D. and Osmond, C.B. (1976) Compartmentation and transport in C, photosynthesis. 1n Transport in Plants. U. Heber and C.R. Stocking, eds., Vol. 3. pp. 145-184. Springer-Verlag, Berlin. Hatch, M.D., Osmond, C.B. and Slatyer, R.O. (Editors) (1971) Photosynthesis and Photorespiration. Wiley-Interscience, New York. Hatch, M.D. and Slack, c.R. (1966) Photosynthesis by sugar cane leaves. A new carboxylation reaction and the pathway of sugar formation. Bioche:m. J. 101: 103-111. Hatch, M.D. and Slack, c.R. (1967) The participation of phosphoenolpyruvate synthase in photosynthetic CO, fixation of tropical grasses. Arch. Brochem-. Biophys. 129: 224-225. Hatch, M.D. and Slack, c.R. (1968) A neW enzyme for the interconversion of pyruvate and phosphopyruvate and its role in the C, dicarboxylic acid pathway of photosynthesis. Bioche:m. J. 106: 141-146. Hatch, M.D. and Slack, c.R. (1969a) Studies on the mechanism of activation and inactivation of pyr";vate, phosphate dikinase. Bioche:m. J, 112: 549-558. Hatch, M.D. and Slack, c.R. (1969b) NADP-specific malate dehydrogenase and glycerate kinase in leaves and their location in chloroplasts. Biochem. Biophys. Res. Commun. 34: 589-593. C4 Photosynthesis 195 Hatch, M.D. and Slack, c.R. (1970a) The C. dicarboxylic acid pathway of photosynthesis. In Progress in Phytochemistry. L. Reinhold and Y. Liwschitz, eds., Vol. 2., pp. 35-106. Wiley Interscience, London. Hatch, M.D. and Slack, c.R. (1970b) Photosynthetic CO, fixation pathways. Annu. Rev. Plant Physioi. 21: 141-162. Hatch, M.D. Slack, c.R. and Bull, T.A. (1969) Light induced changes in the content of some " . em;ymes of the C. dicarboxylic acid pathway of photosynthesis. Phyto-chemistry 8: 697-706. Hatdlt M.D., Slack, c.R. and Johnson, H.S. (1967) Further studies on a new pathway of photbsynthetic CO, fixation in sugar cane and its occurrence in other plant species. Biochem. J. 102: 417-422. Hattersley, P.W. and Watson, L. (1992) Diversification of photosynthesis. In Grass Evolution and Domestication. GP. Chapman, ed., pp. 38-116. Cambridge University Press, Cambridge, UK. Henderson, S.A., von Caenunerer, S. and Farquhar, G.D. (1992) Short-term measurements of carbon isotope discrimination in several C. species. Aust. J. Plant Physiol. 19: 263-285. Hesketh, J.D. (1963) Limitations to photosynthesis responsible for differences among species. Crop Sci. 3: 493-496. Hesketh, J.D. and Moss, DN. (1963) Variations in the response of photosynthesis to light. Crop Sci. 3: 107-110. Hodge, A.J., McLean, J.D. and Mercer, F.V. (1955) Ultrastructure of the lamellae and grana in the chloroplasts of Zea mays L. J. Biophys. Biochem. Cytol. 25: 605-614. Jenkins, C.L.D., Furbank, R.T. and Hatch, M.D. (1989) Mechanism of C, photosynthesis: A model describing the inorganic carbon pool in bundle sheath cells. Plant Physiol. 91: 1372-1381. Johnson, H.S. and Hatch, M.D. (1968) Distribution of the C, dicarboxylic acid pathway and its occurrence in dicotyledonous plants. Phytochemistry 7: 375-380. Johnson, H.S. and Hatch, M.D. (1970) Properties and regulation of leaf NADP-malate dehydrogenase and malic enzyme in plants with the C. dicarboxylic acid pathway of photosynthesis. Biochem. J. 119: 273-280. Karpilov, Yu. S. (1960) The distribution of radioactive carbon 14 amongst the products of photosynthesis of maize. Trudy Kazansk Sel'shokhoz Institute 41(1): 15-24. Karpilov, Yu. S. (1969) Peculiarities of the functions and structure of the photosynthetic apparatus in some species of plants of tropical origin. Proc. Moldavum Inst. Irrigation and Vegetable Research 11: 1-34. Kortschak, H.P., Hartt, C.E. and Burr, GO. (1965) Carbon dioxide fixation in sugar cane leaves. Plant Physiol. 40: 209-213. Kortschak, H.P. and Hartt, C.E. (1966) The effect of varied conditions on carbon dioxide fixation in sugar cane leaves. Naturwissenschaften 53: 253. Laetsch, W.M. (1968) Chloroplast specialisation in dicotyledons possessing the C, dicarboxylic acid pathway of photosynthetic CO, fixation. Am: J. Bot. 55: 875-883. Laetsch, W.M. (1971) Chloroplast structural relationships in leaves of C, plants. In Photosynthesis and Photorespiration. M.D. Hatch, C.B. Osmond and RO. Slatyer, eds., pp. 323-348. Wiley-Interscience, New York. Laetsch, W.M. and Price, I. (1968) Development of the dimorphic chloroplasts of sugar cane. Amer. J. Bot. 56: 77-87. Loomis, R.S. and Williams, W.A. (1963) Maximum crop productivity - an estimate. Crop Sd. 3: 67-72. Marino, B.D., McElroy, M.B., Salawitch, R.J. and Spaulding, W.G (1992) Glacial to interglacial variation in the carbon isotope composition of atmospheric CO,. Nature 357: 461-466. Meidner, H. (1962) The minimum intercellular space CO, concentration of maize leaves and its infl1,lence on stomatol movement. J. Exptl. Botany. 13: 284-293. 196 M, D. Hatch and C. R. Slack Moss, D.N. (1962) The limiting carbon dioxide concentration for photosynthesis. Nature 193: 587. Murata, Y. and lyama, I. (1963) Studies on the photosynthesis of forage crops II. Influence of air . temperature. Proc. Crop. Sci. Soc. Japan 31: 15-322. Nobel, P.S. (1991) Achievable productivities of CAM plants: basis for high values compared with C, and C, plants. New Phytol. 119: 183-205. Osmond, CB. (1967) [3-Carboxylation during photosynthesis in Atriplex. Biochim. Biophys. Acta 141: 197-199. Osmond, CB. and Holtum,. I.A.M. (1981) Crassulacean acid metabolism. In Biochemistry of Plants. VoL 8. Photosynthesis. M.D. Hatch and N.K. Boardman, eds., pp. 283-328. Academic Press, New York. Osmond, CB., Troughton, I.H. and Goodchild, D.I. (1969) Physiological, biochemical and struct1.:tral studies of photosynthesis and photorespiration in two species of Atriplex. Zeit. Pflanzenphysiol. 61: 218-237. Rawsthome, S. (1992) C,-C, intermediate photosynthesis: linking physiology to gene expression. Plant J. 2: 267-274. Rhoades, MM. and Carvalho, A. (1944) The function and structure of the parenchyma sheath plastids of maize leaf. Bull Torrey Botanical Cub 7: 335-346. Shantz, H.L. and Piemeisel, L.N. (1927) The water requirement of plants at Akron Colorado. J. Agr. Res. 34: 1093-1189. Slack, CR. (1968) The photoactivation of phosphopyruvate synthase in leaves of Amaranthus palmerii Biochem. Biophys. Res. Cmnmun. 30: 483-488. Slack, CR. (1969) Localisation of certain photosynthetic enzymes in mesophyll and parenchyma sheath chloroplasts of maize and Amaranthus palmeri. Phytochemistry. 8: 1387-1391. Slack, CR. and Hatch, M.D. (1967) Comparative studies on the activities of carboxylases and other enzymes in relation to the new pathway of photosynthetic CO, fixation in tropical grasses. Biochem. J. 103: 660-665. Slack, CR., Hatch, M.D. and Goodchild, D.]. (1969) Distribution of enzymes in mesophyll and parenchyma sheath chloroplasts of maize leaves in relation to the C, dicarboxylic add pathway of photosynthesis. Biochem. J. 114: 489-498. Smith, BN. (1972) Natural abundance of the stable isotopes of carbon in biological systems. Bioscience 22 : 226-231. Tarchevskii, lA. and Karpilov Yu.S. (1963) On the nature of products of short-term photosynthesis. Soviet Plant Physiol. 10: 183-184. Ting. LP. (1985) Crassulacean acid metabolism. Annu. Rev. Plant Physiol. 36: 595-622. Tregunna, B.B., Smith, B.N., Berry, ].A. and Downton, W.J.S. (1970) Some methods for studying the photosynthetic taxonomy of the Angiosperms. Can. J. Bot. 48: 1209-1214. Webster, R.D. (1987) The Australian Paniceae. Kramer,Berlin and Stuttgart.