Dual Temporal Role of Plastid Sigma Factor 6 in Arabidopsis Development

Heike Loschelder,Gerhard Link,Brigitte Link,Jennifer Schweer

doi:10.1104/pp.106.085878

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.

Highlights

Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription
Sequencing of T-DNA borders identified the insertion site within exon 5 of the genomic Sig6 sequence on chromosome II (At2g36990) 1,022 nucleotides downstream of the ATG initiation start codon, which would correspond to a derived protein that lacks all functional domains for sigma factor activity (Fig. 1A)
We have characterized a new AtSig6 mutant allele, sig6-2, both at the DNA and RNA level, as well as by complementation with intact cDNA. This reverse-genetics strategy established a causal link between the introduced gene and the visual and molecular phenotype of the rescued transformants, both of which resembled that of the wild type (Fig. 5)

Summary

Introduction

Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription. PEP has major roles in functional chloroplasts, which is reflected by the large number of regulatory proteins surrounding the catalytic core (Pfannschmidt et al, 2000; Loschelder et al, 2004; Suzuki et al, 2005; Pfalz et al, 2006), including those that associate transiently, such as the sigma factors (Tiller and Link, 1993a, 1993b). Whereas basic sigma functions have been tested to a large extent using heterologous in vitro systems with authentic plastid or bacterially expressed sigma proteins and Escherichia coli RNA polymerase (Hakimi et al, 2000; Hanaoka et al, 2003; Homann and Link, 2003), the availability of Arabidopsis sigma mutant lines has facilitated functional studies in vivo (Hanaoka et al, 2003; Privat et al, 2003; Nagashima et al, 2004; Tsunoyama et al, 2004; Favory et al, 2005; Ishizaki et al, 2005). We investigate an Arabidopsis knockout line with a Sig mutant allele (Rosso et al, 2003) that reveals a strong developmental stagespecific (albino) phenotype and characteristic changes in plastid gene expression

Methods

Results

Conclusion