Functional analysis of plastid-encoded genes

Plastid chromosomes from the variety of plant species contain
several conserved open reading frames of unknown function, which
most probably represent functional genes. The primary aim of this
thesis was the analysis of the role of two such ORFs, designated
ycfs or hypothetical chloroplast reading frames, namely ycf9
(ORF62) and ycf10 (ORF229, cemA). Both were analyzed in Nicotiana
tabacum (tobacco) via their inactivation using biolistic plastid
transformation. A new experimental protocol, based on pulsed-field
gel electrophoresis (PFGE), was established to reliably assess the
homoplastomic state of transformed plants. 1. Functional analysis
of the ycf9 gene product: The inactivation of ycf9 in N. tabacum as
well as in Chlamydomonas reinhardtii yielded a homoplastomic mutant
phenotype after several rounds of regeneration under selective
pressure. The mutant plants grew photoautotrophically, but
displayed two clear phenotypes, a light-sensitive one, increasing
with the light intensity, and a dwarf phenotype under low-light
combined with temperatures below 20°C. The ycf9 gene product was
exclusively located in PSII core complexes. This localization was
based on the isolation of protein complexes released from
thylakoids by controlled, partial lysis, followed by sucrose
density gradient centrifugation or 2D gel electrophoresis. This
finding revised data of the literature. Biochemical analysis
indicated an involvement of the protein in the interaction of the
light harvesting antenna II complex (LHCII) with PSII cores. In
particular, PSII-LHCII supercomplexes could no longer be isolated
from transplastomic tobacco plants. Furthermore, the minor
chlorophyll a/b-binding proteins CP26, and to a lesser extent CP29,
were substantially reduced under most growth conditions analyzed,
in both, tobacco and photoautotrophically grown Chlamydomonas
mutants (Swiatek et al. 2001). The gene was therefore renamed psbZ.
The ∆psbZ-related alterations in the supramolecular organization of
PSII complexes were accompanied by considerable modification in (i)
the phosphorylation pattern of PSII subunits, (ii) the rate of
deepoxydation of xanthophylls, and (iii) the kinetics and amplitude
of non-photochemical quenching. The proposed position of PsbZ in
close proximity to CP43 enables the protein to interact with PSII
cores to elicit an adaptation process in response to excess light
excitation. The molecular mechanism underlying this energy
dissipation process remains to be investigated. 2. Functional
analysis of the ycf10 gene product: Biolistic plastid
transformation was also used to inactivate the ycf10 reading frame
in tobacco. After several rounds of regeneration under selective
pressure, homoplastomic plants were obtained. Northern analysis
uncovered co-transcription of ycf10 within the psaI-ycf4-ycf10-petA
gene cluster, with at least two promotor regions upstream of the
psaI gene. The mutant plants grew photoautotrophically and
developed dark green leaves with numerous pale green to white
regions, the latter devoid of photosynthetic activity. The loss of
ycf10 did not affect photosynthetic activity, as indicated by
unaltered chlorophyll fluorescence. The tobacco ycf10 gene product
was localized in the chloroplast inner envelope membrane. Neither
protein composition of stroma or thylakoid fractions, nor the
stability of the photosynthetic protein complexes were affected in
the mutant plants. In contrast, CO2- dependent oxygen evolution was
strongly reduced, with a maximum rate of Ci-dependent
photosynthesis being approximately 50% lower than in wild-type
plants. Two explanations can account for the observed phenomenon:
(i) de-regulation of carbonconcentrating mechanisms in transformed
cells, or (ii) an indirect effect on CO2-uptake in ∆ycf10 plants.
3. Pulsed-field gel electrophoresis is an ideal tool to verify the
homoplastomic state of transformed plants: To enhance the
sensitivity of detection of heteroplastomic states, and to
distinguish between plastome-located wild-type segments in
transplastomic material and promiscuous DNA, a new approach was
developed. Customary Southern and PCR techniques are not sensitive
enough or not discriminating the latter alternatives, respectively.
Pulsed-field gel electrophoresis allows to isolate virtually
contamination-free plastid DNA. Plastid DNA isolated this way
lacked traces of nuclear and mitochondrial DNA at a detection level
of 50 DNA molecules. This excludes that gene-specific PCR
amplification products originate from promiscuous nuclear or
mitochondrial gene copies. Therefore, PFGE appears to be an ideal
tool to investigate the homoplastomic state of transformed plants,
especially when combined with radiolabeled probes and Southern
techniques.