Possible Involvement of the 5′-Flanking Region and the 5′UTR of Plastid accD Gene in NEP-Dependent Transcription

Abstract

In many developmentally and functionally important higher plant plastid genes, expression depends on a specific nuclear-encoded RNA polymerase (NEP). Molecular mechanisms for NEP-mediated gene expression are poorly understood. We have improved a transient expression assay based on biolistics and the dual-luciferase reporter technique, which facilitated investigations into the regulation of plastid genes in vivo. We scrutinized the 5'-flanking region and the 5'-untranslated region (5'UTR) of accD, a plastid gene encoding a subunit of the prokaryotic-type acetyl-CoA carboxylase which is transcribed exclusively by NEP. The results indicated that two AT-rich sequences, one of them containing two overlapping YRTA-like motifs, were essential for accD expression in vivo. The results also revealed that the length of the 5'UTR rather than a particular sequence element was a determinant for the level of accD expression. Because transcripts accumulated in proportion to reporter enzyme activity and protein levels, and transcript degradation rates were independent of the nature of the 5'UTR, it was unlikely that the 5'UTR acts as a translational enhancer or a stabilizer of the transcripts. Therefore, the length of 5'UTR might be a factor contributing to the efficiency of NEP-dependent transcription in plastids.

Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome

“…In both cases, underaccumulation of the ncRNA correlated with accumulation of the sense strand. As rbcL and accD are transcribed from individual PEP and NEP promoters, respectively, the intergenic region detected is likely an extension of an rbcL precursor rather than an unprocessed dicistron (Gruissem and Zurawski 1985; Hirata et al 2004). PNPase and RNase E appear to have a role in rbcL maturation that leads to degradation of the intergenic region, as this transcript is not detectable in the WT.…”

Genome-Wide Analysis of Plastid Gene Expression in Potato Leaf Chloroplasts and Tuber Amyloplasts: Transcriptional and Posttranscriptional Control

“…Interestingly, several other essential genes (clpP, ycf1, and ycf2; Drescher et al, 2000;Kuroda and Maliga, 2003) were relatively highly expressed in potato tubers. In transient assays, the length of the accD 5# untranslated region appeared to determine the expression level of a reporter gene in both nonphotosynthetic and photosynthetically competent tobacco plastids (Hirata et al, 2004). Stable transformation experiments are needed to confirm these results for potato amyloplasts and to evaluate the potential of the 5# and 3# regions from accD and the other genes found to be actively expressed in tubers to enhance (trans)gene expression in nongreen plastids.…”

The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera , subsection Oenothera : I. Sequence evaluation and plastome evolution †

“…In tobacco, spinach, Arabidopsis and maize, this cluster is co-transcribed from a PEP promoter upstream of rbc L into a giant RNA species of ∼9 kb that is subsequently processed, often rapidly (Figure 5; 92). Removal of rbc L from the operon either required the replacement of the rbc L promoter by that of trn Q, use of the operon-internal NEP promoter in front of acc D (93), acquisition of a new PEP promoter 5′ to acc D (or trn Q) or use of the NEP promoter upstream of rpo B (c.f. Figure 2).…”