AtSig5 Is an Essential Nucleus-Encoded Arabidopsis σ-Like Factor

Abstract

Transcription of chloroplast genes is subject to control by nucleus-encoded proteins. The chloroplast-encoded RNA polymerase (PEP) is a eubacterial-type RNA polymerase that is presumed to assemble with nucleus-encoded -factors mediating promoter recognition. Recently, families of -factor genes have been identified in several plants including Arabidopsis. One of these genes, Arabidopsis SIG5, encodes a -factor, AtSig5, which is phylogenetically distinct from the other family members. To investigate the role of this plant -factor, two different insertional alleles of the SIG5 gene were identified and characterized. Heterozygous mutant plants showed no visible leaf phenotype, but exhibited siliques containing aborted embryos and unfertilized ovules. Our inability to recover plants homozygous for a SIG5 gene disruption indicates that SIG5 is an essential gene. SIG5 transcripts accumulate in flower tissues, consistent with a role for AtSig5 protein in reproduction. Therefore, SIG5 encodes an essential member of the Arabidopsis -factor family that plays a role in plant reproduction in addition to its previously proposed role in leaf chloroplast gene expression.Transcription of plant mitochondrial and plastid genomes relies on nucleus-encoded RNA polymerases resembling those of the T3 and T7 bacteriophage, as well as promoter selectivity factors for these enzymes (for review, see Hess and Bö rner, 1999;Liere and Maliga, 2001). In addition, plastids, but not mitochondria, require an organelle-encoded RNA polymerase (PEP) for transcription of many genes on the organelle genome (Allison and Maliga, 1996;Hajdukiewicz et al., 1997;Serino and Maliga, 1998). PEP is similar in structure to Escherichia coli RNA polymerase and, like the eubacterial enzyme, is thought to assemble with -factors to achieve promoter-specific transcription initiation. Although the subunits of the PEP catalytic core are plastidencoded, the putative promoter-specificity -factors for PEP are encoded in the plant nucleus. Therefore, even the organelle-encoded transcription machinery is subject to nuclear control.Recently, -factor gene families containing as many as six members have been identified in several plant species including Arabidopsis and maize (Zea mays; for review, see Allison, 2000). Within a species, the sigma-like proteins share approximately 35% overall amino acid sequence identity and contain the conserved domains found in the principal -factors of all eubacteria (Helmann and Chamberlin, 1988). Genetic analyses of the eubacterial -factors, as well as more recent structural studies, have supported the importance of these conserved domains in such eubacterial transcription functions as promoter recognition, interaction with the RNA polymerase core enzyme, and transcription initiation (Gross et al., 1998;Burgess and Anthony, 2001;Campbell et al., 2002;Murakami et al., 2002). The conservation of primary structure among plant and eubacterial -factors is sufficiently high to permit recombinant plant -factors to function with E. coli core RNA po...

ANK6, a mitochondrial ankyrin repeat protein, is required for male-female gamete recognition in Arabidopsis thaliana

“…The heterozygous mutant (+/SIG5-3), but not homozygous mutant, was obtained and displayed a white shriveled ovule phenotype (Fig. 6 B, a), which is similar to the phenotype reported earlier with +/sig5-1 and +/sig5-2 (26). Further analysis of the mutant showed that when normal ovules developed to the early globular embryo stage, a fraction of sig5 ovules, like some in +/ank6 plants, were arrested at the FG1 stage (Fig.…”

“…3B). SIG5 is a member of the σ-factor family of proteins that function as transcription initiation factors in both chloroplasts and mitochondria (26). Interestingly, disruption of SIG5 function was reported to cause homozygous lethality similar to the phenotype of ank6 observed here.…”

“…Interestingly, disruption of SIG5 function was reported to cause homozygous lethality similar to the phenotype of ank6 observed here. Thus, siliques in heterozygous mutant plants (+/sig5) exhibited aborted embryos and unfertilized ovules (26), suggesting that SIG5, like ANK6, plays a role in fertilization or embryo development.…”

“…3 A and B). Although Yao et al (26) detected SIG5 protein in both the reproductive and vegetative organs by immunoblot analysis, a detailed expression pattern of SIG5 in the reproductive organ was not reported. We thus conducted a GUS reporter assay using transgenic plants harboring SIG5 promoter fused to GUS.…”

“…The full-length mature SIG5 has an N-terminal nonconserved region and C-terminal-binding domains (26). To identify the ANK6-interacting domain, six truncated SIG5 variants were fused with GAL4 activation domain (GAD) (Fig.…”