Download The Discovery of C4 Photosynthesis

The Discovery of C4
Photosynthesis
By the end of the 1950s it was widely believed that all plants used the same pathway of
photosynthesis. This pathway was called the Calvin cycle or photosynthetic carbon reduction
(PCR) cycle and began with the incorporation of carbon dioxide (CO 2) into the 5-carbon sugar
ribulose bisphosphate (RuBP) to form a six-carbon intermediate which immediately splits to
form two C3 molecules of 3-phosphoglycerate. In the latter half of the next decade Marshall
Hatch and Roger Slack with, PhD students Hilary Johnson and John Andrews, working at the
Brisbane laboratory of the Colonial Sugar Refining Co Ltd., provided evidence for the
mechanism, enzymology and regulation of an alternative photosynthetic process operating in
leaves of sugarcane. In this process, the first products of CO2 fixation are the C4dicarboxylic
acids, malate, aspartate and oxaloacetate, not the C3 molecule 3-phosphoglycerate. They
named the process the C4 dicarboxylic pathway of photosynthesis, abbreviated later to the
C4 pathway or C4 photosynthesis. Several other grass species and some species from other
families were shown to have a similar mechanism. This alternative process, the NADP-malic
enzyme-type, is one of three types of C4 photosynthesis subsequently identified in later years.
With the closure of the CSR Brisbane laboratory in 1970, Marshall Hatch took up a position
with the CSIRO Division of Plant Industry where he continued studies on this process until his
retirement in 1997. Major contributions during that time included aspects of the resolution of
the mechanism of two alternative biochemical options for C4 photosynthesis, details of the
regulation of these processes and their role as CO2 concentrating mechanisms, identification
of new enzymes, details of the regulation of key enzymes and their intracellular location and
studies on the fluxes of metabolites between cells. He and colleagues finally identified all but
one of the ten major enzymes involved in C4 photosynthesis. Marshall Hatch received many
honours and awards for this work including election as a Fellow of the Royal Society, a Fellow
of the Australian Academy of Science and a Foreign Associate of the National Academy of
Sciences (USA).
The Calvin cycle ‘ the classic pathway of photosynthesis
The 1961 Nobel Prize in Chemistry was awarded to the Russian-born, American scientist
Melvin Calvin from the University of California, Berkeley, for his discovery of the metabolic
processes involved in photosynthesis, the assimilation of carbon dioxide in plants. During the
1950s, Calvin and his colleagues, using C14-radiolabelled CO2, had elucidated the principal
reactions by which the green algaChlorella synthesises glucose from carbon dioxide and
water, using the energy of light. These reactions were termed the Calvin Cycle. Later, they
showed that similar steps, with similar enzymes, occurred in a few higher plants. So, by the
end of the 1950s it was reasonably assumed that this process, termed the Calvin cycle or
photosynthetic carbon reduction (PCR) cycle, accounted for CO2 assimilation in all
photosynthetic organisms.
There are three phases of the Calvin cycle. In phase 1 (Carbon Fixation), CO 2 is incorporated
into a five-carbon sugar named ribulose bisphosphate (RuBP) to form a six-carbon
intermediate which immediately splits in half to form two molecules of 3-phosphoglycerate.
The enzyme which catalyzes this first step is RuBP carboxylase or rubisco. It is the most
abundant protein in chloroplasts and probably the most abundant protein on Earth. In phase 2
(Reduction), ATP and NADPH2 produced from the chlorophyll-catalysed light reactions are
used to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, the three-carbon
carbohydrate precursor to glucose and other sugars via the six terminal enzymatic steps of
gluconeogenesis. In phase 3 (Regeneration), more ATP is used to convert some of the pool
of glyceraldehyde 3-phosphate back to ribulose bisphosphate, the acceptor for CO 2, thereby
completing the cycle.
As summarised in the figure below, for every three molecules of CO2 that enter the cycle, the
net output is one molecule of glyceraldehyde 3-phosphate (G3P). For each G3P synthesized,
the cycle spends nine molecules of ATP and six molecules of NADPH 2.
Figure 1. The Calvin Cycle. One molecule of glucose is generated from 6 molecules of CO2.
Source: Images of the Calvin Cycle –
http://cnx.org/resources/722dad908853869fdd3c65bf0fc65a46
The light reactions sustain the Calvin cycle by regenerating the ATP and NADPH 2 required.
This is donevia two light-harvesting units (photosystems I and II) located in the thylakoid
membranes of chloroplasts.
Early evidence for a deviant
In the early 1960s, while working at the Colonial Sugar Refining Company’s David North Plant
Research Centre in Brisbane, Marshall Hatch and his colleague Roger Slack, through their
interactions with Hugo Kortschak and colleagues at the Hawaiian Sugars Planters Research
laboratory in Honolulu, became aware of the latter’s unpublished observations that
photosynthesis in sugar cane might be more complex. As early as the late 1950s, just a few
years after Calvin’s pioneering studies, the Hawaiian group had seen some unusual patterns
of labelling in products formed when sugarcane leaves were allowed to assimilate 14CO2 in the
light. In contrast to Calvin’s observations of early labelling of 3-phosphoglycerate (3-PGA), the
Hawaiian workers did not find a great deal of label in 3-PGA. Instead much of the early label
was located in the 4 carbon dicarboxylic acids malate and aspartate. Nothing of this was
published for several years. As Marshall Hatch recalled:
‘Roger Slack and I were intrigued by this data from the Hawaiian laboratory and had often
discussed the possible interpretations of these results. So when Kortschak and his colleagues
finally published their data in 1965 we set about repeating and extending their observations to
see if we could resolve the question of what it all meant’.
Hatch went on to state: ‘In the late 1960s, that is 3 or 4 years after we had begun studying the
C4photosynthetic process, we became aware of a report, published about ten years earlier by
a young Russian scientist Y. Karpilov in the 1960 Annual Report of a Russian Agricultural
Research Institute. He found that when maize (Zea mays) leaves were allowed to
assimilate 14CO2 in the light for short periods, most of the fixed label appeared in malate plus
aspartate. Only a small percentage was found in 3-PGA. In a publication about three years
later, Karpilov and a colleague speculated that such results may be related to faulty killing or
extraction procedures, and there the matter rested’.
Young and mature sugarcane crop, Atherton. QLD Photographer : Willem van Aken
The C4 dicarboxylic acid pathway of photosynthesis is
described
Hatch and Slack confirmed the results of the Hawaiian group, that most of the radioactivity
incorporated after short periods in 14CO2 was located in malate and aspartate and that these
C4 acids were rapidly labelled from zero time whereas steady rates of labelling of 3-PGA,
sugar phosphates and sucrose occurred only after increasing lag periods. More revealing and
critical information was provided by their‘pulse-chase‘ experiments where, after a period of
labelling in 14CO2, leaves are transferred back to air containing unlabelled CO2 and the
movement of fixed radioactivity between compounds is followed. These experiments clearly
showed the rapid movement of radioactivity from malate into 3-PGA and then later into
hexose phosphates and other intermediates and finally into sucrose and starch (Figure 3).
Other critical results reported in these initial studies included the findings that

the chemically unstable dicarboxylic acid, oxaloacetate, was rapidly labelled as well
as malate and aspartate and that oxaloacetate was almost certainly the first product
formed,

the C4 acids were initially labelled almost entirely in the C-4 carboxyl,

this C4 carboxyl carbon gave rise to the C-1 carboxyl of 3-PGA,

3-PGA was converted to hexose phosphates apparently by the same path as normal
Calvin cycle photosynthesis, and

a survey of a large number of plants showed labelling patterns similar to sugarcane in
14 grass species from 10 genera as well as a sedge species.
As a result of these initial studies, Hatch and Slack developed a simple working model to
explain photosynthesis in sugarcane. In this model they proposed that in the initial reaction, a
3-carbon compound, probably phosphoenolpyruvate (PEP) or pyruvate, was carboxylated to
give a C4 dicarboxylic acid with the C-4 carboxyl derived from CO2. After reactions
interconverting the C4 acids, oxaloacetate, malate and aspartate, the 4-carboxyl of one of
these acids is transferred to become the carboxyl group of 3-PGA and they speculated that
the remaining 3-carbons of the dicarboxylic acid might serve as a precursor to regenerate
pyruvate or PEP. Soon after, they named this process the C4 dicarboxylic acid pathway of
photosynthesis which was later abbreviated to the C4 pathway or C4 photosynthesis (see
Figure 3).
Figure 3. C4 photosynthesis
Figure 3. C4 photosynthesis is an evolutionary development where specialised mesophyll
cells initially fix CO2from the air into 4-carbon acids which are transported to the site of the
Calvin (PCR) cycle in the bundle sheath. The bundle sheath cells are relatively impermeable
to CO2, so that when the CO2 is released here from the 4-carbon acids, it builds up to high
levels. The C4 photosynthetic mechanism is a biochemical CO2 pump. (a) An abbreviated
scheme for C4 photosynthesis in relation to C4 leaf anatomy. C4 photosynthesis is an
evolutionary pathway shown here overlayed on a micrograph of a C 4 leaf, showing bundle
sheath and mesophyll cells. Rubisco and the other PCR enzymes are in the bundle sheath
cells while phosphoenolpyruvate (PEP) carboxylase is part of the CO2 pump in the mesophyll
cells. (b) A schematic representation of pulse-chase labelling in C4 leaves showing the
transfer of radioactivity from C4 acids initially to 3-PGA and then to photosynthetic endproducts following transfer to air containing unlabelled CO2. Scale bar = 10 µm. (Original
drawings courtesy M.D. Hatch).
Then followed a series of studies where predictions based on existing information led to
discoveries about the enzymes involved which in turn allowed further predictions. For
instance, radiotracer studies predicted a primary carboxylation reaction involving either PEP
or pyruvate. A search for such an enzyme revealed that sugarcane and other plants showing
this unconventional C4-type labelling contained very high levels of PEP carboxylase, with up
to 50 times the activities seen in leaves of non-C4-type plants. Later studies led to the
discovery of the novel enzymes, ‘pyruvate, Pi dikinase’ and the ‘NADP-specific malate
dehydrogenase’, and evidence of special roles for several other enzymes including adenylate
kinase, pyrophosphatase, aspartate aminotransferase and NADP malic enzyme. As Marshall
Hatch recalled:
‘It is probably worth mentioning that both pyruvate, Pi dikinase and NADP malate
dehydrogenase turned out to be dark/light regulated, with high activities extractable from
illuminated leaves but virtually no activity recoverable after leaves were held for several
minutes in low light or darkness. You can imagine the difficulties this caused in the early
stages of our studies on these enzymes, with activities varying widely from day to day
depending on how fast one got from the glasshouse to the laboratory. The widely differing
mechanisms for the dark/light regulation of these enzymes was to keep us amused off and on
for the next twenty years or so’.
By 1970, work on photosynthesis in the Colonial Sugar Refining Company laboratory in
Brisbane had been terminated and the group broke up. Roger Slack joined the Department of
Scientific and Industrial Research (DSIR) laboratory in New Zealand and Marshall Hatch
joined the CSIRO Division of Plant Industry in Canberra.
Discoveries at CSIRO
In December 1970, an international meeting on Photosynthesis and Photorespiration was
held in Canberra, and provided the first opportunity for the growing number of people with an
interest in C4photosynthesis to get together. As Marshall Hatch recalled: ‘This was a
marvellously organized meeting thanks mainly to Ralph Slatyer, one of the best I have ever
attended – a meeting where schedules were flexible and there was time for real discussion ‘.
The proceedings of this meeting were published in 1971 and helped to cement together into
some kind of cohesive story, the various strands of the overall C4photosynthetic
phenomenon- the unique biochemistry with the special Kranz anatomy and ultrastructural
features, the special physiological and performance characteristics, and the emerging
taxonomic patterns distinguishing C3 and C4 plants. At this meeting, Olle Bjorkman (from the
Carnegie Institute, Stanford) and Marshall Hatch, proposed what was to become a very
important element of C4philosophy – that the function of the process was to concentrate
CO2 for fixation by the Calvin cycle. Hatch’s evidence a year later – that C4 leaves develop a
large pool of inorganic carbon during photosynthesis – supported this view.
The following five years saw the delineation of two alternatives to NADP malic enzyme for the
decarboxylation of C4 acids in bundle sheath cells. These options were PEP carboxykinase
and NAD malic enzyme and this provided the basis for dividing C 4 plants into three distinct
biochemical sub-types, NADP-malic enzyme-type, NAD-malic enzyme-type and PEP
carboxykinase-type. The fact that evolving C4 plants came up with three separate
mechanisms for decarboxylating C4 acids in bundle sheath cells was one of the many
surprising features of this process. Other surprises included the involvement of mitochondria
in C4 acid decarboxylation in two of these three sub-groups and the operation of unique
chloroplasts with no Rubisco and sometimes no Photosystem II activity. There was also the
unprecedented mechanism for dark/light regulation of pyruvate, Pi dikinase (see below). In
reflecting on this time Marshall Hatch wrote in his memoir ‘I can’t believe my luck’:
‘It is inevitable that as one works through a major research project, the most difficult problems
gradually get filtered out and are left to last. For instance, we spent many years trying to
understand the nature of the decarboxylation process in PEP carboxykinase-type species.
Only recently have we got a reasonable understanding of this mechanism, thanks to Jim
Burnell’s perseverance. Understanding the unique mechanism of dark-light regulation of
pyruvate, Pi dikinase was a long and slow process extending over nearly twenty years. In
recent times we have also been able to directly and quantitatively demonstrate what was
suspected for some time – that the mesophyll-bundle sheath cell interface in C4 leaves is
remarkably permeable to metabolites, thus facilitating the essential rapid flux of metabolites
between mesophyll and bundle sheath cells, but remarkably impermeable to CO 2, thus
permitting the concentrating of CO2 in bundle sheath cells with acceptable efficiency. We are
also slowly getting more quantitative information about the bundle sheath inorganic carbon
pool and the likely concentration of CO2, as such, in these cells. However, we still lack a direct
quantitative assessment of the extent that CO2 leaks from bundle sheath cells during
photosynthesis’.
The studies on pyruvate, Pi dikinase were plagued by an array of unusual circumstances and
experimental problems that mitigated against its discovery and the elucidation of its
mechanism of action and regulation. Early studies on the enzyme were difficult because of its
ephemeral activity. It was rapidly inactivated when leaves were transferred to low light or
darkness, and the extracted enzyme rapidly lost activity in the absence of Mg 2+, unless a thiol
such as dithiothreitol was present, or if it was maintained at a temperature below 10 °C’.
As Marshall Hatch concluded: ‘Periodic bursts of experimental activity over many years finally
revealed that both the inactivation and activation processes are mechanistically unique.
Inactivation results from phosphorylation of a threonine residue by ADP, but only if a catalytic
site histidine is already phosphorylated. This is a remarkable reaction since, except for the
adenylate kinase reaction, Nature seems to have otherwise studiously avoided the potential
of ADP as a phosphosphorylating agent. The reasons remain unclear. P ireactivates the
enzyme by a phosphorolytic cleavage of the threonine-P to give PPi – also a unique reaction
as a mechanism for enzyme dephosphorylation. To cap it off, these two quite different
reactions are catalysed by the same enzyme which we termed the pyruvate, P i dikinase
regulatory protein, a very rare situation’.
Figure 4. Dr MD (Hal) Hatch, FAA, FRS, co-discoverer of C4 photosynthesis.
Source:http://plantsinaction.science.uq.edu.au/content/feature-essay-21-discovery-c4photosynthesis.
How common is C4 photosynthesis?
Plants with the C4 pathway are called C4 plants to distinguish them from C3 plants which use
only the Calvin cycle for photosynthesis. An estimate in 1999 (Sage RF et al., 1999, In: C4
Plant Biology, Sage RF, Monson RK, eds, pp551-584), predicted that there are between
8,000 ‘ 10,000 species of C4 plants in ~500 genera. They occur in two monocotyledonous
families ‘ Gramineae and Cyperaceae ‘ (6,000 ‘ 8,000 species in ~400 genera) as well as
several dicotyledonous families (2000 species in 100 genera).
C4 plants are capable of higher rates of leaf photosynthesis than C3 plants, especially at
higher temperatures, show higher water-use efficiency and are commonly more tolerant to
drought. They grow more rapidly and produce more dry matter than C 3 plants under
appropriate conditions. These featrures can be explained in terms of their capacity to
concentrate carbon dioxide for use by the Calvin cycle. The resolution of the mechanism and
function of C4 photosynthesis has been important in understanding the molecular basis of
plant productivity.
Evolution of C4 photosynthesis
About 100 million years ago C3 plants were in their ‘prime’ with atmospheric
CO2 concentrations between five and ten times present day levels. However, a new selection
pressure then developed. Atmospheric CO2 declined over the next 50’60 million years to
something close to our twentieth century levels of about 350 µL ‘1. This decline almost
certainly provided the driving force for evolution of C 4photosynthesis. In other words,
C4 photosynthesis was originally ‘discovered’ by nature in the course of overcoming the
adverse effects of lower atmospheric CO2 concentration on C3 plants. In effect, C4processes
increase the CO2 concentration in bundle sheath cells to somewhere near the atmospheric
CO2 concentration of 100 million years ago.
Approximately 85% of all terrestrial plant species perform C3 photosynthesis, while about 3%
fix atmospheric CO2 via the C4 photosynthetic pathway. About 10% of plants carry out
crassulation acid metabolism (CAM) and are usually found in highly xeric sites (deserts,
epiphytic habitats). Less common photosynthetic modes are single-cell C4, C3-C4 intermediate
and SAM photosynthesis the latter found insubmerged aquatic macrophytes such as pond
weeds and seagrasses.
Ribulose bisphosphate carboxylase (rubisco) is a surprisingly inefï¬cient enzyme with a slow
turnover of active sites and a rather feeble discrimination between alternative substrates
(CO2 and O2), a combination that severely restricts photosynthetic performance of C 3 plants
under ambient conditions of 20% O2 and 0.035% CO2. Accordingly, and in response to
CO2 limitation, C4, CAM and SAM variants have evolved with metabolic concentrating devices
which enhance Rubisco performance.
C4 plants predominate in open and arid habitats, and also include several important food
crops such as maize and sugarcane. C4 plants have a competitive advantage over C3 plants
at high temperature and under strong light because of a reduction in photorespiration and an
increase in absolute rates of CO2fixation at current ambient CO2. Such increase in
photosynthetic efficiency results in faster carbon gain and commonly higher growth rates,
particularly in subtropical and tropical environments. Consequently, and in response to the
looming food security crisis, a global research effort led by IRRI (International Rice Research
Institute) is underway to bioengineer C4 photosynthetic traits into major C3 crops, such as rice,
in order to boost their photosynthesis, and thus, improve yield and resource use efficiency.
Sources

Hatch MD, 2015, Personal communication

Hatch MD, 1992, ‘I can’t believe my luck’, Photosynthesis Research, 33: 1-14.

Plants In Action, second
edition, http://plantsinaction.science.uq.edu.au/content/chapter-2-carbondioxide-assimilation-and-respiration

International Prize for Biology citation at https://www.jsps.go.jp/english/ebiol/data/list/7th-ipb_en.pdf