No abstract

Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription. The in vivo function of individual members of the sigma gene family has become increasingly accessible by knockout and complementation strategies. Here we have investigated plastid gene expression in an Arabidopsis (Arabidopsis thaliana) mutant with a defective gene for sigma factor 6. RNA gel-blot hybridization and real-time reverse transcription polymerase chain reaction together indicate that this factor has a dual developmental role, with both early and persistent (long-term) activities. The early role is evident from the sharp decrease of certain plastid transcripts only in young mutant seedlings. The second (persistent) role is reflected by the up-and down-regulation of other transcripts at the time of primary leaf formation and subsequent vegetative development. We conclude that sigma 6 does not represent a general factor, but seems to have specialized roles in developmental stage-and gene-specific plastid transcription. The possibility that plastid DNA copy number might be responsible for the altered transcript patterns in mutant versus wild type was excluded by the results of DNA gel-blot hybridization. Retransformation of the knockout line with the full-length sigma 6 cDNA further established a causal relationship between the functional sigma gene and the resulting phenotype.

Chloroplasts sigma factors act in concert with PEP, the bacterial-type plastid RNA polymerase. Using a sigma knockout line from Arabidopsis thaliana, we investigated mutant-specific changes in plastid gene expression at RNA level. One characteristic feature was the appearance of a long transcript that spans the atpB-E operon and extends considerably into the far-upstream region of atpB. This region reveals a cluster of typical promoter elements for NEP, the second (phagetype) plastid RNA polymerase. The NEP promoter cluster can help maintain RNA synthesis in situations where no functional sigma factor is available for PEP.

Plastids contain sigma factors, i.e. gene-regulatory proteins for promoter binding and transcription initiation. Despite the physical and functional similarity shared with their prokaryotic counterparts, the plant sigma factors have distinguishing features: most notably the existence of a variable extra sequence comprising their N-terminal portions. This distinct architecture is reflected by functional differences, including phosphorylation control by organellar protein kinase(s) closely related to nucleocytosolic, rather than bacterial-type, enzymes. In particular, cpCK2, a nuclear-coded plastid-targeted casein kinase 2, has been implicated as a key component in plant sigma factor phosphorylation and transcriptional regulation (Eur. J. Biochem. 269, 2002, 3329; Planta, 219, 2004, 298). Although this notion is based mainly on biochemical evidence and in vitro systems, the recent availability of Arabidopsis sigma knock-out lines for complementation by intact and mutant sigma cDNAs has opened up new strategies for the study of transcription regulatory mechanisms in vivo. Using Arabidopsis sigma factor 6 (AtSIG6) as a paradigm, we present data suggesting that: (i) this factor is a substrate for regulatory phosphorylation by cpCK2 both in vitro and in vivo; (ii) cpCK2 phosphorylation of SIG6 occurs at multiple sites, which can widely differ in their effect on the visual and/or molecular phenotype; (iii) in vivo usage of the perhaps most critical cpCK2 site defined by Ser174 requires (pre-)phosphorylation at the n + 3 serine residue Ser177, pointing to ‘pathfinder’ kinase activity capable of generating a functional cpCK2 substrate site.

Like bacteria, plastids contain sigma factors for promoter binding and transcription initiation. Accumulating evidence suggests that members of the plant sigma factor family can have specialized non-redundant roles in terms of promoter preference in various developmental and environmental situations. To specify regulatory determinants, we have chosen pairwise exchange of portions of Arabidopsis sigma coding regions, followed by transformation of the chimeric constructs into a sigma 6 knockout line. The resulting phenotypes and plastid RNA patterns point to an important though not exclusive role for the highly variable N-terminal portion of plant sigma proteins.