The role of thylakoid ATP synthase subunit gamma and attempts to transform the organelles of A. thaliana

ATP synthase is one of the major photosynthetic complexes that
represents one of the smallest molecular motors known in nature.
The rotating γ subunit is a key feature of this enzyme. It contains
features specific for the chloroplast ATP synthase. In this work
the γ subunit has been functionally analyzed in Arabidopsis
thaliana. - The nuclear gene atpC1 encoding the γ subunit of the
plastid ATP synthase has been inactivated by T-DNA insertion
mutagenesis. In the seedling-lethal dpa1 mutant the absence of
detectable amounts of the γ subunit destabilizes the entire ATP
synthase complex and consequently photophosphorylation is
abolished. However, in vivo protein labelling analysis suggests
that assemαβ bly of the ATP synthase and subunits into the
thylakoid membrane still occurs in dpa1. Further effects of the
mutation include an increased light sensitivity of the plants and
an altered photosystem II activity. A high non-photochemical
quenching develops with increasing actinic light intensity. It has
been shown that a high proton gradient is responsible for most
quenching (qE). The photoprotective role of qE was further
demonstrated in the double mutant dpa1 x psbS in which PsbS,
essential factor for qE, is missing. - The expression of a second
gene copy, atpC2, is unaltered in dpa1 and is not sufficient to
compensate for the lack of atpC1 expression. The two proteins,
AtpC1 and AtpC2, share less similarity than AtpC1 of Arabidopsis
with γ subunits of other plant species suggesting that the γ
subunits so far isolated in other plant species are AtpC1
orthologs. It has been established that AtpC2 is also imported into
the chloroplast. Therefore, it is likely that the chloroplast ATP
synthase complexes contain both atpC1 and atpC2 encoded γ subunits.
However, the atpC2 gene is expressed more than hundred times at a
lower level than atpC1 and array data show the differential and
tissue specific expression of the two genes. The function of AtpC2
could not be revealed by inactivating the gene. Overexpression of
atpC2 in dpa1 generated viable lines with an ATP synthase complex
containing only γ2, although wild type phenotype is not completely
restored. The second part of this work regarded the optimization of
conditions for plastid transformation in Arabidopsis thaliana. An
efficient and fast regeneration system from cotyledon protoplasts
was established for Arabidopsis thaliana accessions C24, Columbia,
and Wassilewskija. Culture conditions and media compositions were
optimized for the development of protoplasts embedded in thin
alginate layers. The protocol is reproducible, efficient, extremely
fast, and regenerated plants are fertile. Thus, this
cotyledon-based system could prove useful for studying plant cell
and molecular biology in A. thaliana. - The sul gene appeared to be
a potential novel candidate as selectable marker for plastid
transformation. However, genetic and molecular studies demonstrated
that sul can not be used for this purpose. On the other hand a new
function of sul appeared. The gene could be the missing marker for
mitochondria transformation in higher plants.