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TRENDS in Biotechnology
Vol.25 No.10
437
Engineering photorespiration in chloroplasts: a novel
strategy for increasing biomass production
Muhammad Sarwar Khan
National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan
Photosynthetic carbon metabolism is rate limiting in C3
plants because of a competing process: photorespiration. Photorespiration lowers the energy efficiency of
photosynthesis by metabolizing glycolate produced
by the oxygenate activity of Rubisco. The chloroplasts
of Arabidopsis thaliana have recently been reported
to contain a novel respiratory pathway that converts
glycolate directly to glycerate and thus increases productivity by improving photosynthesis in transgenic
plants. This pathway promises to widen the applicability
of the approach to other C3 plants.
Introduction
Photosynthetic carbon metabolism is a key factor in plant
growth and yield. Photosynthetic carbon fixation, catalyzed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is a fundamentally inefficient
process for most plants; this has been extensively reviewed
elsewhere [1–3]. Rubisco works very slowly; it catalyzes
only a few reactions per second. A further limitation on the
efficiency of carbon dioxide fixation is the ability of oxygen
to bind to the active site of the enzyme in a non-productive
reaction in which ribulose bisphosphate is broken down
and carbon dioxide is released; this process is known as
photorespiration [4]. Thus, Rubisco catalyzes two competing reactions, carboxylation and oxygenation, the rates
of which depend upon the relative concentrations of
CO2 and O2, as well as on temperature. Carboxylation
leads to net CO2 fixation, whereas oxygenation generates
glycolate that can only be metabolized outside chloroplasts
by photorespiratory processes in peroxisomes and mitochondria [5]. Therefore, plant growth and yield can be
improved by increased photosynthesis and/or by reduced
photorespiration.
The efficiency of photosynthesis in C3 plants (plants,
e.g. wheat and rice, that fix CO2 in the form of three
carbon-atom molecules) could be improved either if
Rubisco were made more efficient or if its binding specificity for CO2 rather than for oxygen were increased.
Furthermore, prospecting for more-efficient variants
and concentrating CO2 in the vicinity of the enzyme by
introducing the C4 pathway of photosynthesis could
improve the photosynthetic efficiency of C3 plants. In C4
plants (plants, e.g. maize and sugarcane, that fix CO2 in
the form of four carbon-atom molecules), enhanced CO2
fixation is caused by the coordination of two cell types:
Corresponding author: Khan, M.S. ([email protected]).
Available online 17 September 2007.
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mesophyll and bundle sheath cells. The arrangement of
these cells around the vascular tissue, referred to as Kranz
anatomy [6], differs in C4 plants, as does the mechanism of
transport of metabolites between these cells [6]. Nevertheless, all C4 plants initially fix HCO3 by using phosphoenolpyruvate (PEP) carboxylase to form oxaloacetate
in the cytoplasm of mesophyll cells (Figure 1a). CO2 enters
mesophyll cells and is converted to HCO3 by carbonic
anhydrase. Finally, the CO2 librated during decarboxylation of malate, the product of oxaloacetate, is fixed by
Rubisco in the chloroplasts of neighboring bundle sheath
cells [6]. However, in Hydrilla verticillata, a single-cell
monocot that acclimates to declining CO2 by shifting from
C3 to C4 photosynthesis [7], the librated CO2 is fixed in the
same cell (Figure 1b).
The discovery of an alternate version of Rubisco that
would improve the efficiency of photosynthesis has long
been the ‘Holy Grail’ of plant biology, but despite considerable effort, this aim has yet to be realized. Engineering C3
plants with the C4 pathway seems to be more promising,
although some have questioned the benefits of concentrating CO2 in the chloroplasts of C3 plants because they are
known to leak gases [8].
Recently, Kebeish et al. [9] outlined a novel approach to
alleviating photorespiratory losses in Arabidopsis thaliana; this approach reduced photorespiration and enhanced
photosynthesis by the release of CO2 in the vicinity of
Rubisco. The approach is based on incorporating a bacterial pathway for the catabolism of the photorespiratory
substrate, glycolate. Engineered chloroplastic photorespiration increases photosynthesis and biomass and thus
promises to widen the applicability of this approach to C3
plants.
Photorespiration and the photorespiratory pathway
in C3 plants
Photorespiration is the metabolism of phosphoglycolate
that is produced during oxygenation catalyzed by
Rubisco. Photorespiration inhibits photosynthesis by
interfering with CO2 fixation catalyzed by Rubisco.
Furthermore, it lowers energy efficiency by metabolizing
phosphoglycolate produced by Rubisco. Energy efficiency
is lowered further by the absence of a CO2-concentrating
mechanism in C3 plants [4]. Overall, in photorespiratory
events one molecule of CO2 is released for every two
molecules of phosphoglycolate produced, a net loss of
fixed carbon that reduces the production of sugars and
biomass. Ammonia is also released by this reaction and
needs to be ‘refixed’ by energy-consuming reactions in the
chloroplasts [2].
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TRENDS in Biotechnology Vol.25 No.10
Figure 1. Schematic representation of C4 photosynthesis. (a) Photosynthesis in plants with Kranz anatomy. The CO2 enters the cytoplasm of mesophyll cell and is converted
into HCO3 by carbonic anhydrase. The phosphoenolpyruvate (PEP) carboxylase fixes bicarbonate to produce oxaloacetate, which then enters the chloroplasts. The C4 acid
is then converted into malate by malate dehydrogenase. The decarboxylation of malate is catalyzed by malate enzyme to generate pyruvate, thus releasing CO2 in
chloroplasts of bundle sheath cells. The pyruvate diffuses back to chloroplasts of mesophyll cells, where it is converted into PEP by pyruvate orthophosphate dikinase to
continue the CO2 fixation cycle. (b) Photosynthesis in a single-cell monocot, such as Hydrilla verticillata, that lacks Kranz anatomy but acclimates to declining CO2 with a
shift from C3 to C4 photosynthesis. All steps involved in CO2 fixation are the same as in C4 plants; however, these steps are carried out in the same cell rather than in two
different cells.
Photorespiration is a seemingly wasteful reaction that
adversely affects photosynthesis in C3 plants. However, it
protects plants from high light intensities by playing a
critical role in dissipating excess photochemical energy and
thus protecting chloroplasts from over-reduction [10,11]. A
variety of photorespiratory mutants of A. thaliana are able
to grow in a high-CO2 environment but not at ambient CO2
concentrations, highlighting the importance of photorespiration in C3 plants [12]. Nevertheless, high CO2 levels
favor photosynthesis by suppressing the photorespiratory
reaction, supporting the notion that suppression of photorespiration in C3 plants could improve photosynthesis
under similar environmental conditions.
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Engineering chloroplast-based photorespiration in
C3 plants
A pathway for the catabolism of a photorespiratory
substrate, glycolate, has been reported for Escherichia coli
[13,14] and plants [15]. In E. coli, glycolate dehydrogenase
(GDH) uses NAD+ as an electron acceptor to oxidize glycolate to glyoxylate. However, in plants, glycolate is oxidized to glyoxylate by glycolate oxidases that use molecular
oxygen in peroxisomes. Nevertheless, a glycolate pathway
similar to the recently engineered photorespiratory bypass
in A. thaliana was reported for Synechocystis sp. strain
PCC 6803 [16]. The role of the normal photorespiration
pathway in cyanobacteria is not yet clear.
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TRENDS in Biotechnology
Vol.25 No.10
439
Figure 2. Engineered photorespiratory pathway in Arabidopsis thaliana. The Escherichia coli glycolate catabolic pathway is introduced to chloroplasts by step-wise
incorporation of genes into the nuclear genome and post-translational targeting of proteins to the chloroplasts. The pathway works independently in chloroplasts.
Carboxylation of 5-bisphosphate (RuBP) by Rubisco generates glycerate-3-phosphate for use within the Calvin cycle, whereas oxygenation generates glycolate-2-phosphate
and glycerate-3-phosphate. Colored arrows and metabolites indicate the glycolate catabolic pathway in chloroplasts, whereas black arrows and metabolites in
peroxisomes, mitochondria and chloroplasts depict endogenous plant photorespiration. Abbreviations: glyoxylate carboligase (GCL); glycine decarboxylase (GDC);
glycolate dehydrogenase (GDH); ribulose1,5-bisphosphate (RuBP); ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); tartronic semialdehyde reductase (TSR).
Taking advantage of a fundamental difference between
bacterial and plant glycolate metabolic enzymes, Kebeish
et al. [9] established the pathway in chloroplasts by stepwise nuclear transformation and post-translational targeting of proteins to plastids (Figure 2). Engineering the
alternate photorespiratory pathway in chloroplasts was
conceived based on the observation that the growth of
barley and A. thaliana mutants deficient in photorespiration remained unaffected by high CO2 concentrations.
However, under low CO2 concentrations the growth of
mutants was drastically reduced, supporting the general
perception that partial suppression of photorespiration
might not be detrimental to C3 plants.
To establish the glycolate catabolic pathway in
chloroplasts, Kebeish et al. constructed three plasmid
vectors for the transformation of Arabidopsis. The three
vectors harbored five genes encoding subunits of GDH,
GCL and TSR, respectively, tethered to the constitutive
35S promoter of cauliflower mosaic virus and potato-chloroplast-targeting sequence. The authors first targeted the
three subunits of glycolate dehydrogenase (GDH) to chloroplasts and then introduced glyoxylate carboligase (GCL)
and tartronic semialdehyde reductase (TSR) to complete
the competitive photorespiratory pathway for converting
glycolate to glycerate. In sequential transformations, two
independent transgenic lines were produced; one (designated as the DEF line) carried genes for three subunits (D,
E and F) of GDH and the other (designated as the GT line)
contained genes encoding GCL and TSR. The DEF line was
produced by super-transformation of a transgenic line
expressing subunit F with a vector carrying genes for
the D and E subunits. Crossing DEF and GT plants
provided the complete pathway.
Expression of the novel pathway in chloroplasts results in
a reduction of photorespiratory flow and, in turn, an increase
in CO2 concentration in the vicinity of Rubisco; this
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enhances carboxylation relative to oxygenation. The
postillumination CO2 burst (PIB) is a measure of the CO2
released by the mitochondrial glycine decarboxylase reaction, during which glycine is converted to serine. A reduction
of 30% in the measured PIB levels in DEF and GT-DEF
lines in comparison to wild-type plants confirms the reduced
photorespiratory flux in transgenic lines.
Plant growth measurements revealed that transgenic
plants expressing the glycolate pathway in their chloroplasts have a larger leaf area and an increased rosette
diameter in comparison to control plants. Moreover,
measurements of total fresh and dry weight showed that
total plant productivity was enhanced. Interestingly, most
of the described effects were also observed in plants that
only overexpressed a functional GDH. However, the effects
were stronger in plants overexpressing all necessary
elements of the glycolate pathway.
Biochemical, physiological and biophysical analyses
were performed under ambient and enhanced photorespiratory conditions (low CO2 concentrations) so that the
impact of the novel pathway in planta could be evaluated.
The findings demonstrated that the transformants have
enhanced rates of carbon fixation, growth and biomass
production at low CO2 concentrations, but no differences
in growth were observed in plants grown under high CO2
concentrations. Nevertheless, an increase in biomass was
evident under stress conditions of high temperature or
intense light, which increase photorespiration. Furthermore, increased levels of soluble sugars in plants that
expressed the bacterial glycolate catabolic pathway in
their chloroplasts but had no differences in Rubisco content
indicate that these plants have a greater biomass. Interestingly, the reduction in the CO2 compensation point
of the transgenic plants compared to wild-type plants
provides further evidence for the physiological significance
of the novel biochemical pathway in C3 plants.
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TRENDS in Biotechnology Vol.25 No.10
Conclusion
The distinguishing characteristic of the recently reported
photorespiratory pathway in chloroplasts is that, unlike
the endogenous photorespiratory pathway, it generates
energy while eliminating the utilization of ATP and
thus conserving power that is normally required to refix
ammonia in the glutamine synthetase/glutamate synthase
cycle. Engineered chloroplastic photorespiration increases
photosynthesis and biomass and thus promises to widen
applicability of the approach to other C3 crops, such as
wheat, rice and particularly cotton, that are normally
grown under conditions of stress. Nevertheless, further
investigation into how intermediates of the novel pathway
affect the basal transport system and the regulation of
various metabolic processes in chloroplasts is required.
Precisely how glycolate oxidation in the chloroplasts
improves biomass production of field-grown crops under
variable growth conditions requires further research. A
possible improvement to the system is the use of regulated
promoters that can induce expression of genes that encode
the novel photorespiratory pathway under low-photosynthesis conditions. Another point that should be considered
when transferring the pathway to C3 crops is that the plant
can become loaded with several undesired marker genes;
this could be avoided if the pathway were transferred into
chloroplasts in the form of an operon [3].
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0167-7799/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tibtech.2007.08.007
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