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Molecular Plant Breeding 2012, Vol.3, No.9, 91
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high-density metals like tungsten and gold particles
are coated with DNA and propelled into the target
cells through acceleration provided by high pressure
helium gas as propellant. It was experienced that
tungsten and gold particles work equally well, but,
oxidized layer on tungsten particles is removed before
coating DNA. In most laboratories, biolistic delivery
method is preferred over other methods to develop
transplastomic plants because unlike polyethylene
glycol (PEG) treatment of protoplasts, fully expanded
leaves, cotyledons, or cultured cells in tissue culture
are used (Svab and Maliga, 1993; Khan and Maliga,
1999; Ruf et al., 2001, G. Mustafa and M.S. Khan,
unpublished). Further, PEG-treatment of protoplasts is
restricted to tobacco only. Alternate methods for DNA
delivery to plastids have also been tried. Microinjection,
which looks promising for transient gene expression
(Knoblauch, et al., 1999), has not yet yielded stable
transplastomic clones.
2 Functional analyses of plastid genes using
omics approaches
The complete nucleotide sequence of an ever-increasing
number of plastid genomes have revealed that they are
remarkably conserved in their coding capacity and
organization in higher plants and algal species. The
genome organization and plastid gene expression has
been described elsewhere in detail (Daniell and Khan,
2003). Plastids have their own transcription and
translation machineries (Gruissem and Tonkyn, 1993),
which were conserved through the evolutionary steps.
The availability of complete genome sequences, a
large number of expressed sequence tags and
introduction of high-throughput methodologies such
as transcriptomics and proteomics has enabled the
analyses of gene expression in photosynthetic organisms
from transcription to post-translational levels, and to
study the protein-protein interactions (Duggen et al
1999; Cahoon and Timko, 2000; Yamamoto et al.,
2009; Armbruster et al., 2011). DNA microarray
technology has been used successfully to functionally
analyze the gene expression at transcriptomes level in
various plants (Schaffer et al., 2000). Further, ‘RNA-seq’,
a deep sequencing approach has been successfully
used to investigate the transcriptional control of
ectopic chloroplast development in plants, considering
the approach a powerful tool to compare gene
expression on a genome-wide scale in a species,
without a reference genome (Zhou et al., 2011).
Recently, transplastomic tomato transcriptomes during
fruit development and chloroplasts to chromoplasts
conversion are analyzed (Kahlau and Bock, 2008).
When compared with leaves, most but not all genes
were strongly down regulated in fruits. However,
such large changes in RNA accumulation during
chloroplast-to-chromoplast differentiation at the time of
fruit ripening were not observed; perhaps most plastid
genes were translationally down regulated. It is well
documented that the transcriptional activity varies from
one gene to another as a result of its important role in
plastid differentiation and the control of gene
expression in response to environmental cues. The
transcription in plastids is largely dependent on nuclear
genes, encoding polymerases, δ factors and various
other transcription regulators (Shiina et al., 2005).
Customized DNA microarrays have also been
developed for studying photosynthesis related gene
expression profiles (Kurth et al., 2002) because
genes with similar functions often display similar
transcriptional profiles. Lately, multiple approaches
have been used to characterize genes, for example,
‘guilt-by-association’ concept is coupled with reverse
genetic approach and a number of genes of unknown
functions that exhibit photosynthesis genes like
transcription profile are characterized (Dalcorso et al.,
2008). Genome-wide characterization of
psaA
and
psbA
genes deletion mutants using transcriptomic and
quantitative proteomic analyses have revealed that
the nuclear and chloroplast genes involved in
photosynthesis, energy metabolism and chloroplast
biogenesis, and genes transcribed by PEP are down
regulated whereas genes transcribed by the nuclear
encoded polymerase (NEP) are up-regulated (Khan et
al., 2007; Leelavathi et al., 2011), indicating simultaneous
activation of multiple signaling pathways in response
to disruption of PSI and PSII complexes in Δ
psaA
and
Δ
psbA
plants (Leelavathi et al., 2011). Hence, these
studies have paved the path to study functional
analysis of plastid–nuclear interactions.
However, from proteomics perspective, two-dimensional
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