Control Of Plastid Gene Expression Research Articles

H4 acetylation by the NuA4 complex is required for plastid transcription and chloroplast biogenesis

Chloroplast biogenesis is crucial in plant development, as it is essential for the transition to autotrophic growth. This process is light-induced and relies on the orchestrated transcription of nuclear and plastid genes, enabling the effective assembly and regulation of the photosynthetic machinery. Here we reveal a new regulation level for this process by showing the involvement of chromatin remodelling in the nuclear control of plastid gene expression for proper chloroplast biogenesis and function. The two Arabidopsis homologues of yeast EPL1 protein, components of the NuA4 histone acetyltransferase complex, are essential for plastid transcription and correct chloroplast development and performance. We show that EPL1 proteins are light-regulated and necessary for concerted expression of nuclear genes encoding most components of chloroplast transcriptional machinery, directly mediating H4K5ac deposition at these loci and promoting the expression of plastid genes required for chloroplast biogenesis. These data unveil a NuA4-mediated mechanism regulating chloroplast biogenesis that links the transcription of nuclear and plastid genomes during chloroplast development.

The cutting crew - ribonucleases are key players in the control of plastid gene expression

Chloroplast biogenesis requires constant adjustment of RNA homeostasis under conditions of on-going developmental and environmental change and its regulation is achieved mainly by post-transcriptional control mechanisms mediated by various nucleus-encoded ribonucleases. More than 180 ribonucleases are annotated in Arabidopsis, but only 17 are predicted to localize to the chloroplast. Although different ribonucleases act at different RNA target sites in vivo, most nucleases that attack RNA are thought to lack intrinsic cleavage specificity and show non-specific activity in vitro. In vivo, specificity is thought to be imposed by auxiliary RNA-binding proteins, including members of the huge pentatricopeptide repeat family, which protect RNAs from non-specific nucleolytic attack by masking otherwise vulnerable sites. RNA stability is also influenced by secondary structure, polyadenylation, and ribosome binding. Ribonucleases may cleave at internal sites (endonucleases) or digest successively from the 5' or 3' end of the polynucleotide chain (exonucleases). In bacteria, RNases act in the maturation of rRNA and tRNA precursors, as well as in initiating the degradation of mRNAs and small non-coding RNAs. Many ribonucleases in the chloroplasts of higher plants possess homologies to their bacterial counterparts, but their precise functions have rarely been described. However, many ribonucleases present in the chloroplast process polycistronic rRNAs, tRNAs, and mRNAs. The resulting production of monocistronic, translationally competent mRNAs may represent an adaptation to the eukaryotic cellular environment. This review provides a basic overview of the current knowledge of RNases in plastids and highlights gaps to stimulate future studies.

External control of transgene expression in tobacco plastids using the bacterial lac repressor

Although several induction systems have been described for plants containing transgenes in the nucleus, to date there is only one method for controlling transgene expression in plastids. This consists of chemical induction of a nuclear gene and import of the gene product into plastids, so that transformation of two cellular compartments is required. Here we describe a system for external control of plastid gene expression which is based entirely on plastid components and can therefore be established in a single transformation step. Our system uses modified promoters containing binding sites for the bacterial lac repressor. Chemical induction can be made with intact plants or after harvesting, which provides ecological and economic benefits.

Mutations in a gene for plastid ribosomal protein S6-like protein reveal a novel developmental process required for the correct organization of lateral root meristem in Arabidopsis.

Arabidopsis rfc3 mutants have previously been isolated with an altered fatty acid composition of membrane lipids. In this study, rfc3 was found to have a sucrose-conditional defect in the patterning of distal elements in the lateral root meristem. By utilizing this feature, a sucrose-sensitive process important for lateral root development was localized to the growing portion of rfc3 primary root. Because lateral root formation occurs at a later stage, this finding suggests the existence of an RFC3-dependent, non-primordium autonomous signal playing a role in the organization of lateral root meristem. Map-based cloning of RFC3 gene revealed that it encodes a plastid-localized ribosomal protein S6-like protein and provides a potential link between control of plastid gene expression and LR development.

PEG-mediated plastid transformation in higher plants

Plastids are surrounded by an envelope consisting of a double membrane. This barrier has to be penetrated or overcome by the DNA when transforming the plastome. Both the biolistic and polyethylene glycol-mediated transformation techniques accomplish this task, albeit by different mechanisms. We were the first laboratory to successfully use the polyethylene glycol (PEG)-method for plastid transformation, yet we use the particle gun when appropriate. In this report we compare the two methods and discuss their shortcomings and advantages. Plastid transformations with various constructs, mainly using theaadA gene as a selective marker, were performed. We point to potential problems likely to be encountered during the transformation and selection processes and offer possibilities for improvement. We give further examples of the successful application of plastome transformation and show its merits in addressing biological questions concerning the elucidation of plastid sequences of unknown function and the control of plastid gene expression.

Transcriptional control of plastid gene expression during development of primary foliage leaves of barley grown under a daily light-dark regime

Plastid DNA transcription was studied during biogenesis and maturation of chloroplasts in primary foliage leaves of barley (Hordeum vulgare L.) seedlings grown under a daily light-dark regime. Specific age-dependent changes in the transcript pattern of plastids have been shown by hybridization to barley plastid DNA fragments of run-on transcript probes derived from homogeneous populations of proplastids and chloroplasts of different age. In proplastids, transcription of the rrn operon is predominant and the increasing transcriptional activity during biogenesis of chloroplasts is mainly the consequence of additional transcription of mRNA and tRNA genes. During maturation of chloroplasts, rrn transcription preferentially decreases and, conversely, transcription of the psbD gene increases. The importance of these changes in plastid DNA transcription is discussed in relation to chloroplast ageing.

Transcriptional control of plastid gene expression in greening Sorghum seedlings

The greening of 5-d-old etiolated Sorghum seedlings was used to investigate regulatory mechanisms of plastid gene expression. Sorghum was selected for this study because leaves of etiolated seedlings do not expand in the dark and are kept within the coleoptile, thus leaf development is inhibited during etiolation. Only traces of thylakoid-membrane polypeptides and plastid transcripts could be detected in dark-grown seedlings, the accumulation of both protein and RNA was found to be strictly dependent upon light. Run-on transcription analysis demonstrated that the rise in plastid RNA levels was due to a light-induced increase in plastid transcriptional activity and not to altered RNA stability. Using an antiserum to the β subunit of plastid RNA polymerase, it could be demonstrated that the increase in plastid transcriptional activity was paralleled by rising levels of plastid RNA polymerase. It has to be concluded, therefore, that in greening Sorghum seedlings the expression of plastid genes is photoregulated at the transcriptional level.

Light-induced transformation of etioplasts to chloroplasts of barley without transcriptional control of plastid gene expression

Transcription in etioplasts and chloroplasts of 5 day old barley plants was compared by hybridization of corresponding plastid run-on transcripts to cloned DNA fragments of small size and in sum representing the complete plastid genome. Hybridization signals reflecting the relative transcription rates of the various DNA fragments were almost identical in etioplasts and chloroplasts. Parallel determinations of the relative steady state concentrations of plastid RNA species showed no correlation between synthesis rate and accumulation of transcripts. Differences in the relative steady state levels of RNAs in etioplasts and chloroplasts were hardly detectable. The results indicate that in barley light-dependent transformation of etioplasts to chloroplasts may proceed without obvious changes in the rate of transcription or the stability of transcripts of plastid genes.

Expression of plastid and nuclear genes during chromoplast differentiation in bell pepper (Capsicum annuum) and sunflower (Helianthus annuus)

The expression of genes involved in plastid differentiation is subject to developmental control. To understand better the molecular basis of this regulation, we have analyzed in parallel the changes in relative levels of four chloroplast and two nuclear transcripts during differentiation of chloroplasts into chromoplasts in ripening bell pepper fruits and in maturing sunflower petals, as well as in the case of chemically-induced chromoplast formation in leaves. Amongst the prominent features, we noted that transcripts of nuclear genes coding for chloroplast proteins, such as the major chlorophyll a/b-binding protein (encoded by cab) and the small subunit of ribuloses-1,5-bisphosphate carboxylase (encoded by rbcS), were detected in chromoplast-containing bell pepper leaves and sunflower petals, whilst they disappeared in bell pepper fruit chromoplasts. Transcripts of the plastid gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase were detected during the whole differentiation process. The steady-state level of the mRNA corresponding to the 32 kDa herbicide-binding protein increased in fruit and petal chromoplasts. Changes in the relative transcriptional activities of plastid genes and in their relative mRNA levels occurred during bell pepper fruit ripening. However, no significant change in the overall transcriptional activity was found when comparing both types of plastids, whereas dramatic changes in translational activity occur, indicating the prevalence of translational control of plastid gene expression in the chromoplast differentiation process.

Control of plastid gene expression: 3' inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription.

We have examined the function of inverted repeat sequences found at the 3' ends of plastid DNA transcription units in higher plants, using a homologous in vitro transcription extract. The inverted repeat sequences are ineffective as transcription terminators, but serve as efficient RNA processing elements. Synthetic RNAs are processed in a 3'-5' direction by a nuclease activity present in the transcription extract, generating nearly homogeneous 3' ends distal to the inverted repeat sequence. S1 nuclease protection experiments demonstrate that the 3' ends generated in vitro coincide with those found for plastid mRNAs in vivo. RNA molecules possessing inverted repeats near their 3' ends are substantially more stable than control RNAs in the chloroplast extract, and kinetic measurements indicate that each RNA has a unique decay rate. Coupled with previously published information suggesting that the differential accumulation of plastid RNAs during development is effectively controlled by post-transcriptional mechanisms, these results raise the possibility that RNA processing and stability, specifically involving 3' end inverted repeats, are important regulatory features of plastid gene expression.

Control of plastid gene expression during development: The limited role of transcriptional regulation

We have analyzed the transcriptional regulation of plastid genes during chloroplast development in illuminated spinach cotyledons and during leaf formation. The RNAs encoded by plastid genes accumulate with different kinetics during the developmental transitions. Using a novel plastid run-on transcription assay we demonstrate that the transcriptional regulation of a large, diverse group of chloroplast genes is of relatively minor importance for the control of their expression. The general transcriptional activity of the plastid genome increases after illumination and decreases during leaf development. This modulation of general transcriptional activity affects most plastid genes simultaneously and is not correlated with adjustments of the plastid DNA copy number. There are no major changes in the relative transcriptional activities of different genes, although their steady-state mRNA levels change dramatically. The analysis of ten specific plastid genes shows that their relative transcriptional activities are largely maintained throughout the developmental program. This limited transcriptional regulation suggests that plastid gene expression in higher plants is effectively controlled at the posttranscriptional level.

The complexity of gene expression in higher plants

During light-induced chloroplast formation in higher plants the synthesis of several nuclear encoded plastid proteins is under the control of phytochrome. Light acting through the phytochrome system is able both to increase the transcription of certain nuclear genes and to decrease the transcription of others. It has been generally assumed that regulation by phytochrome alone would be sufficient to account for the observed light-dependent changes in nuclear gene expression during chloroplast formation. However, it has recently become evident that the light-dependent control of nuclear gene expression may be far more complex than originally expected. There are at least two other factors that in addition to phytochrome may affect nuclear gene expression: (1) changes in chromatin organization from an inactive to a transcriptionally active state, and (2) a plastid-derived factor that seems to be involved in the transcriptional control of some nuclear genes encoding plastid-specific proteins. Although the light-dependent control of transcription has been studied intensively for nuclear genes, much less is known about the light-dependent control of plastid gene expression. The P700 chlorophyll a protein of photosystem I is a major membrane protein whose massive accumulation is induced by light and whose genes have been located on the plastid DNA. In barley a high concentration of mRNA for the P700 chlorophyll a protein was detected within the total RNA as well as within the polysomal fraction of etioplasts and remained almost constant during greening. Based on these results it can be inferred that the accumulation of the P700 chlorophyll a protein during light-dependent chloroplast development in barley is not coupled to its transcript concentration but is controlled at a translational — or post-translational - level. The possible function of protochlorophyllide as photoreceptor in this light-dependent control of plastid gene expression is discussed.