Download Discoveries in Plant Biology 1998 Hatch

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
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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
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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
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i
Hexose phosphates
diphosphate 1)
carboxyl acceptor
3C
3-Phospho- )
glyc~rate .
:
~bulose
2~
.
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IC·
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Aspartate
'hO(:'::I1~~a,. }
3C
1
2C+C*02
1
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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
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z
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PYRUVATE-rALANINE ASPARTATE
p~~~e::7:A::.t:;~-N:2~
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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.
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