Studies of transposon-induced nuclear mutations that affect chloroplast gene expression and assembly
--Alice Barkan, Kirsten Munck, Fenella Raymond, and Rodger Voelker
To elucidate mechanisms by which nuclear genes control chloroplast gene expression and assembly, studies are being conducted with a collection of nuclear mutants defective in chloroplast biogenesis. Twenty-seven hcf mutants have been recovered from the Mu stocks propagated by the Hake and Freeling groups in Berkeley. These mutants are slightly pale green, non-photosynthetic, and (consequently) seedling lethal. At least fifteen of these twenty-seven mutants exhibit the late somatic reversion events typical of Mu-induced mutations. Results of Western analyses indicate that ten mutants lack primarily a single thylakoid membrane complex: three lack specifically the chloroplast ATP synthase, one lacks PSII, four lack PSI, and two lack the cytochrome f/b6 complex. With the exception of the PSII mutant (see contribution by Voelker and Barkan), these phenotypes may result from lesions in genes encoding a structural component of the affected complex; we have, therefore, not studied them in detail. The remaining seventeen mutants are more certain to define regulatory or assembly functions since multiple photosynthetic complexes are affected. These mutations may define genes that regulate the expression of sets of nuclear or chloroplast genes, that are involved in protein targeting, or that mediate the assembly of multimeric complexes.
To begin to distinguish between these possibilities, we have analyzed the chloroplast transcripts encoding proteins that fail to accumulate, and the association of these transcripts with polysomes. Results of these studies indicate that four mutants (hcf129, hcf131, hcf133, and hcf146) exhibit reduced rates of translation initiation in the chloroplast, in that all plastid mRNAs are associated with abnormally few ribosomes. Nonetheless, the ribosomal RNAs are normal in size and abundance. The genes defined by these mutations are therefore likely to encode components of the chloroplast translation machinery.
Three mutants exhibit clear and unique alterations in chloroplast mRNA metabolism. hcf136 is defective in the endonucleolytic cleavage event that separates the petB and petD coding regions of the polycistronic primary transcript. No other aberrations in RNA processing have been detected in this mutant. The petB and petD gene products are completely missing in hcf136, suggesting that the endonucleolytic cut that fails to occur is essential for generating translatable mRNAs from the primary transcript. hcf143 contains aberrant transcripts of the rps12 and atpF genes. While both of these genes contain class II introns, other genes containing class II introns give rise to normal transcripts, indicating that the defect does not extend to all class II intron-containing genes. The aberrant transcript patterns suggest that splicing still occurs in these mutants, but splicing intermediates and excised introns may overaccumulate. hcf142 fails to accumulate the predominant petA transcript. Many other transcripts accumulate normally. hcf142 therefore has a fairly specific defect in either the transcription or stability of the petA mRNA.
Finally, four mutants that fail to accumulate multiple photosynthetic complexes do not have any detectable defect in chloroplast RNA metabolism or translation (hcf124, hcf137, hcf139, and hcf140). These mutants are therefore likely to be defective in a post-translational aspect of chloroplast biogenesis, such as protein targeting or enzyme assembly. In the future we plan to characterize in more detail these four "assembly" mutants, as well as the three mutants defective in mRNA metabolism. Several of the most interesting mutations will be cloned by taking advantage of the Mu tag.
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