Identification of novel nuclear factors required for chloroplast gene expression and photosystem I assembly

During evolution of photoautotrophic eukaryotes, the nucleus has
gained a dominant role in the coordination of the integrated
genetic system of the cell consisting of three specifically
coevolved genetic compartments. The photosynthetic machinery is
encoded by the chloroplast and nuclear genomes. Therefore,
biosynthesis and assembly of stochiometric amounts of subunits as
well as association of the proteins with corresponding cofactors
need to be managed and precisely regulated. To identify novel
nuclear-encoded factors involved in the regulation of chloroplast
gene expression at different levels, 12 nuclear mutants with high
chlorophyll fluorescence (hcf) phenotypes denoting quite diverse
defects in the photosynthetic apparatus were selected. Three of
them, hcf145, hcf109 and hcf101, were analysed and the affected
genes were characterized in more detail. Spectroscopic,
fluorimetric and immunological studies have revealed that hcf145
and hcf101 were predominantly affected in photosystem I (PSI),
while hcf109 had pleiotropic deficiencies. Remarkably, the dramatic
reduction of PSI core complex accumulation in hcf145 was not
accompanied by corresponding deficiencies of the outer
light-harvesting antenna complex. A comparison of stationary
transcript levels with rates of transcription, as estimated by
Northern and chloroplast run-on transcription analysis, revealed
that the hcf145 mutant is primarily and specifically characterised
by a reduced stability of tricistronic chloroplast psaA-psaB-rps14
transcripts. The corresponding operon encodes the two large PSI
polypeptides PsaA and PsaB, which form the heterodimeric PSI
reaction centre, and the ribosomal protein S14. Chloroplast
translation inhibition experiments excluded translational defects
as the primary cause of impaired mRNA stability. Defined intervals
of the tricistronic transcript were quantified by real-time RT-PCR
which established that the psaA region is less stable than the
rps14 region in hcf145. Therefore, although up to date, no 5'-3'
exoribonucleases have been found in eubacteria (including the
ancestors of plants), factor HCF145 appears to be required for the
protection of the psaA-psaB-rps14 mRNA against progressive
ribonucleolytic degradation starting at the 5' end. In the hcf109
mutant, exclusively plastid transcripts containing UGA stop codons
are unstable. The affected gene encodes the first described
chloroplast peptide chain release factor AtprfB. Its full-length
cDNA, introduced into hcf109 via Agrobacterium-mediated
transformation, could functionally complement the mutant. Homology
of AtprfB to eubacterial release factors indicates that processes
of translational termination in chloroplasts resemble those in
eubacteria. The mutant phenotype revealed that translation of all
plastid mRNAs containing UGA stop codons is exclusively terminated
by AtprfB. However, besides its peptide chain release function,
AtprfB appears to acquire yet unknown roles in regulating the
stability and translation of the chloroplast mRNAs containing UGA
stop codons. These additional regulatory functions could reflect
evolutionary constraints which keep the number of plastid TGA stop
codons high in vascular plant organelles in contrast to those of
algae, mosses and ferns. In contrast to hcf145, steady-state levels
and translation of photosynthetic transcripts are not altered in
the PSI mutant hcf101. Separation of thylakoid membrane complexes
by sucrose gradient centrifugation has uncovered that, similar to
hcf145, accumulation of the outer antenna of PSI is not changed in
hcf101. Therefore, hcf101 is affected in the assembly of the PSI
core complexes. Expression of the HCF101 full-length cDNA in the
hcf101 genetic background functionally complemented the mutant. The
HCF101 protein encodes a very ancient and universally conserved
protein of P-loop ATPases. HCF101 is plastid-localised and
represents the first described factor essentially required for the
assembly of PSI and other [4Fe-4S]-containing protein complexes in
the chloroplast. Relatives of HCF101 are divided into four classes
present in all organisms and in all cellular compartments. The
antiquity of HCF101 points to the importance of Fe-S cluster
biogenesis during the earliest phases of cell evolution. The
ubiquity of HCF101 indicates that it is essential for all
free-living cells.