Lori A. Allison

The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids

The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli‐like RNA polymerase (PEP) which initiates transcription from E.coli σ70‐type promoters. We have previously established the existence of a second nuclear‐encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed exclusively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general mechanism for group‐specific gene expression through recognition by the two RNA polymerases.

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.

The role of sigma factors in plastid transcription

Expression of plastid genes is controlled at both transcriptional and post-transcriptional levels in response to developmental and environmental signals. In many cases this regulation is mediated by nuclear-encoded proteins acting in concert with the endogenous plastid gene expression machinery. Transcription in plastids is accomplished by two distinct RNA polymerase enzymes, one of which resembles eubacterial RNA polymerases in both subunit structure and promoter recognition properties. The holoenzyme contains a catalytic core composed of plastid-encoded subunits, assembled with a nuclear-encoded promoter-specificity factor, sigma. Based on examples of transcriptional regulation in bacteria, it is proposed that differential activation of sigma factors may provide the nucleus with a mechanism to control expression of groups of plastid genes. Hence, much effort has focused on identifying and characterizing sigma-like factors in plants. While fractionation studies had identified several candidate sigma factors in purified RNA polymerase preparations, it was only 4 years ago that the first sigma factor genes were cloned from two photosynthetic eukaryotes, both of which were red algae. More recently this achievement has extended to the identification of families of sigma-like factor genes from several species of vascular plants. Now, efforts in the field are directed at understanding the roles in plastid transcription of each member of the rapidly expanding plant sigma factor gene family. Recent results suggest that accumulation of individual sigma-like factors is controlled by light, by plastid type and/or by a particular stage of chloroplast development. These data mesh nicely with accumulating evidence that the core sigma-binding regions of plastid promoters mediate regulated transcription in response to light-regime and plastid type or developmental state. In this review I will outline progress made to date in identifying and characterizing the sigma-like factors of plants, and in dissecting their potential roles in chloroplast gene expression.

rbcL Transcript Levels in Tobacco Plastids Are Independent of Light: Reduced Dark Transcription Rate Is Compensated by Increased mRNA Stability

The plastid rbcL gene, encoding the large subunit of ribulose-1,5-bisphosphate carboxylase, in higher plants is transcribed from a σ70 promoter by the eubacterial-type RNA polymerase. To identify regulatory elements outside of the rbcL −10/−35 promoter core, we constructed transplastomic tobacco plants with uidA reporter genes expressed from rbcL promoter derivatives. Promoter activity was characterized by measuring steady state levels of uidA mRNA on RNA gel blots and by measuring promoter strength in run-on transcription assays. We report here that the rbcL core promoter is sufficient to obtain wild-type rates of transcription. Furthermore, the rates of transcription were up to 10-fold higher in light-grown leaves than in dark-adapted plants. Although the rates of transcription were lower in the dark, rbcL mRNA accumulated to similar levels in light-grown and dark-adapted leaves. Accumulation of uidA mRNA from most rbcL promoter deletion derivatives directly reflected the relative rates of transcription: high in the light-grown and low in the dark-adapted leaves. However, uidA mRNA accumulated to high levels in a light-independent fashion as long as a segment encoding a stem–loop structure in the 5′ untranslated region was included in the promoter construct. This finding indicates that lower rates of rbcL transcription in the dark are compensated by increased mRNA stability.

Alfalfa Enod12 genes are differentially regulated during nodule development by Nod factors and Rhizobium invasion.

MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa). Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach. MsEnod12A RNA was detected only in nodules and not in other plant tissues. In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves. MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible. In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone. In empty Fix nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable. These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules. Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

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

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 σ-factors mediating promoter recognition. Recently, families of σ-factor genes have been identified in several plants including Arabidopsis. One of these genes, Arabidopsis SIG5, encodes a σ-factor, AtSig5, which is phylogenetically distinct from the other family members. To investigate the role of this plant σ-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 σ-factor family that plays a role in plant reproduction in addition to its previously proposed role in leaf chloroplast gene expression.

Sequences upstream of the YRTA core region are essential for transcription of the tobacco atpB NEP promoter in chloroplasts in vivo

Abstract. Transcription of the plastid atpB gene is accomplished by plastid-encoded (PEP) and nuclear-encoded (NEP) RNA polymerases. In contrast to NEP promoters of many other plastid genes, the tobacco atpB NEP promoter exhibits robust activity in chloroplasts in vivo. Previously, in vitro transcription assays using extracts from non-photosynthetic cells identified two elements required for full atpB NEP promoter activity, a core sequence and an upstream GAA box. Of these, only the core sequence containing the motif YRTA is conserved in the majority of NEP promoters. We used plastid transformation to examine the requirements for atpB NEP promoter activity in chloroplasts. Our results demonstrate that sequences upstream of the core element are essential for promoter activity in vivo, and that transcription of the NEP promoter in chloroplasts is not dependent on activity from an overlapping PEP promoter.

Increased mRNA Stability Compensates for Reduced Dark rbcL Transcription Rates in Tobacco Plastids

The plastid rbcL gene, encoding the large subunit of ribulose-1, 5-bisphosphatecarboxylase, in higher plants is transcribed from a σ70 promoter by the eubacterial-type plastid-encoded plastid RNA polymerase (PEP). In vitro studies have confirmed the importance of the -35 and -10 box spacing and sequence for the rbcL promoter strength (1, 2). Furthermore, sequences positioned between nucleotides -16 and -102 relative to the rbcL transcription initiation site were proposed to function in maize as a binding site for the chloroplast DNA-binding factor 1 (CDF1) sequence-specific transcription factor (3). To identify regulatory elements outside the rbcL -10/-35 promoter core, transplastomic tobacco plants were constructed with uidA reporter genes expressed from rbcL promoter derivatives.