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Molecular Plant Breeding 2012, Vol.3, No.9, 91
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97
shoots for further analysis (Khan et al., 2007).
4 Engineering pathways in chloroplast to
improve photosynthesis
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 (Cleland et al., 1998;
Spreitzer, 1999; Bock and Khan, 2004). Major
limitations on the efficiency of carbon dioxide
fixation are: first, Rubisco works very slowly and it
catalyzes only a few reactions per second, and second,
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 (Wingler et al., 2000). Thus,
Rubisco catalyzes two competing reactions, carboxylation
and oxygenation, the rates of which depend upon the
relative concentrations of CO
2
and O
2
, as well as on
temperature. Carboxylation leads to net CO
2
fixation,
whereas oxygenation generates glycolate that can only
be metabolized outside chloroplasts by photorespiratory
processes in peroxisomes and mitochondria (Medrano
et al., 1995). Therefore, plant growth and yield can be
improved by increased photosynthesis and/or by
reduced photorespiration. Though, the discovery of an
alternate version of Rubisco would improve the
efficiency of photosynthesis but despite considerable
effort, this aim has yet to be realized. Engineering C3
plants with the C4 pathway seems to be more
promising but benefits of concentrating CO
2
in the
chloroplasts of C3 plants have been questioned
because they are known to leak gases (Tolbert, 1997).
Kebeish et al (2007) has incorporated glycolate
catabolic pathway in chloroplasts to alleviating
photorespiratory losses in
Arabidopsis thaliana
. To
establish the glycolate catabolic pathway in
chloroplasts, three vectors harboring five genes that
encode subunits of GDH, GCL and TSR, respectively,
were developed. The authors first targeted the three
subunits of glycolate dehydrogenase (GDH) to
chloroplasts after expressing from nuclear genome
and then introduced glyoxylate carboligase (GCL) and
tartronic semialdehyde reductase (TSR) to complete
the competitive photorespiratory pathway for converting
glycolate to glycerate. Engineered pathway increases
photosynthesis and thus promises to widen
applicability of the approach to cereals, such as wheat
and rice.
Another approach to improving photosynthesis in
plants is: they can develop chlorophyll and assemble
functional chloroplasts in the dark and are ready for
photosynthesis upon exposure to light (Yamazaki et al.,
2006; Kusumi et al., 2006). Chlorophyll biosynthesis is
a multi reactions pathway in which the reduction of
protochlorophyllide (PChlide) to chlorophyllide (Chlide)
that subsequently converts into the chlorophyll by
phytylation is a major regulatory step. The reduction
of Pchlide to Chlide is catalyzed by two different
enzymes: a light dependent, nuclear-encoded, plastid
localized single subunit enzyme (LPOR, Light-dependent
protochlorophyllide oxidoreductase) that requires
light for its activation in angiosperms, and a light
independent plastid-encoded enzyme (DPOR, Dark-
operated protochlorophyllide oxidoreductase) which is
composed of three subunits, encoded by genes
namely,
chlL
,
chlN
and
chlB
. Incorporating DPOR
in the genomes of plastids of cereal crops can improve
photosynthesis by increasing chlorophyll contents,
consequently developing photosynthetically competent
chloroplasts in the dark which will be ready for
photosynthesis upon exposure to light. Recently, the
genes
chlL
,
chlN
and
chlB
from the plastid genome of
Pinus thubergii
, a Japanese Black Pine are introduced
into the plastome of
Nicotiana tabacum
(Nazir, 2012).
The transformed tobacco plants recovered on
spectinomycin-containing regeneration medium were
tested using PCR, showing successful integration of
expression cassettes into the chloroplast genome and
also the homoplasmy was determined through
Southern blot analysis. As these genes are involved in
chlorophyll biosynthesis affecting photosynthetic
performance of the plants, thus homoplasmic transgenic
plants carrying
chlB
genes have been first investigated.
Two exciting observation are made: one, shoots from
the cuttings of the transgenic plants developed early
and more roots in the dark whereas shoots developed
from wild type cuttings showed etiolated growth with
no roots. Upon shifting from dark to light in growth
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