Regulation Of Chloroplast Gene Expression Research Articles

The Chloroplast Epitranscriptome: Factors, Sites, Regulation, and Detection Methods.

Modifications in nucleic acids are present in all three domains of life. More than 170 distinct chemical modifications have been reported in cellular RNAs to date. Collectively termed as epitranscriptome, these RNA modifications are often dynamic and involve distinct regulatory proteins that install, remove, and interpret these marks in a site-specific manner. Covalent nucleotide modifications-such as methylations at diverse positions in the bases, polyuridylation, and pseudouridylation and many others impact various events in the lifecycle of an RNA such as folding, localization, processing, stability, ribosome assembly, and translational processes and are thus crucial regulators of the RNA metabolism. In plants, the nuclear/cytoplasmic epitranscriptome plays important roles in a wide range of biological processes, such as organ development, viral infection, and physiological means. Notably, recent transcriptome-wide analyses have also revealed novel dynamic modifications not only in plant nuclear/cytoplasmic RNAs related to photosynthesis but especially in chloroplast mRNAs, suggesting important and hitherto undefined regulatory steps in plastid functions and gene expression. Here we report on the latest findings of known plastid RNA modifications and highlight their relevance for the post-transcriptional regulation of chloroplast gene expression and their role in controlling plant development, stress reactions, and acclimation processes.

Nuclear-Ecoded Plastid RNA Polymerases Are Components of Anterograde Control in Hormonal Regulation of Chloroplast Gene Expression

Phytohormones are known to play an important role in the regulation of chloroplast biogenesis. However, information on the pathways of hormonal signal perception by chloroplasts is limited. In order to analyze the possible mechanisms of hormone action, the response to abscisic acid (ABA) and cytokinin (CKs) treatment of Arabidopsis thaliana (L.) Heynh. scabra3-2 (sca3-2) and rpotmp mutants with defective genes for nuclear-encoded plastid RNA polymerases was studied. The reaction of the rpotmp mutant usually did not differ from the response of wild-type plants: trans-zeatin activated the accumulation of transcripts of genes encoding the transcription apparatus and genes encoded by the plastid genome, while ABA weakly suppressed their expression or had no significant effect. However, the steady-state levels of gene templates of the plastid-encoded RNA polymerase (PEP-A) enzyme complex in the rpotmp mutant were higher than in wild-type plants, probably due to a compensatory mechanism that allows the mutant to enhance PEP-dependent transcription in the absence of the RPOTmp enzyme. Dysfunction of the plastid RNA polymerase RPOTp enzyme in the sca3-2 mutant induced an altered hormonal response of the genes for plastid transcriptional complex and plastid-encoded genes in comparison with the response of wild-type plants. The absence of a significant up-regulating effect of CKs on the expression of genes encoding the transcription apparatus correlated with the retention of the steady-state levels of transcripts for chloroplast genes and plastid proteins. In addition, the sca3-2 mutant had an increased steady-state expression of genes for CK metabolism: IPT3, IPT5, and CKX5. However, the response to the inhibitory effect of ABA in this mutant, on the contrary, was more pronounced, which is possibly caused by the changes in the steady-state levels of the transcripts of a number of genes encoding of ABA signaling and metabolism proteins. Thus, changes in the expression of NEP (nuclear-encoded RNA polymerases) genes under the action of hormones can be transformed into activation or inhibition in the expression of chloroplast genes of nuclear or plastome coding, providing the regulation of chloroplast functions through the mechanisms of anterograde signaling.

Arabidopsis thaliana egy2 mutants display altered expression level of genes encoding crucial photosystem II proteins

EGY2 is a zinc-containing, intramembrane protease located in the thylakoid membrane. It is considered to be involved in the regulated intramembrane proteolysis – a mechanism leading to activation of membrane-anchored transcription factors through proteolytic cleavage, which causes them to be released from the membrane. The physiological functions of EGY2 in chloroplasts remains poorly understood. To answer the question of what the significance is of EGY2 in chloroplast functioning, two T-DNA insertion lines devoid of EGY2 protein were obtained and the mutant phenotype and photosystem II parameters were analyzed. Chlorophyll fluorescence measurements revealed that the lack of EGY2 protease caused changes in non-photochemical quenching (NPQ) and minimum fluorescence yield (F0) as well as a higher sensitivity of photosystem II (PSII) to photoinhibition. Further immunoblot analysis revealed significant changes in the accumulation levels of the three chloroplast-encoded PSII core apoproteins: PsbA (D1) and PsbD (D2) forming the PSII reaction center and PsbC – a protein component of CP43, a part of the inner PSII antenna. The accumulation levels of nuclear-encoded proteins,Lhcb1-3, components of the major light-harvesting complex II (LHCII) as well as proteins forming minor peripheral antennae complexes, namely Lhcb4 (CP29), Lhcb5 (CP26), and Lhcb6 (CP24) remain, however, unchanged. The lack of EGY2 led to a significant increase in the level of PsbA (D1) with a simultaneous decrease in the accumulation levels of PsbC (CP43) and PsbD (D2). To test the hypothesis that the observed changes in the abundance of chloroplast-encoded proteins are a consequence of changes in gene expression levels, real-time PCR was performed. The results obtained show that egy2 mutants display an increased expression of PSBA and a reduction in the PSBD and PSBC genes. Simultaneously pTAC10, pTAC16 and FLN1 proteins were found to accumulate in thylakoid membranes of analyzed mutant lines. These proteins interact with the core complex of plastid-encoded RNA polymerase and may be involved in the regulation of chloroplast gene expression.

Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation.

Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization, and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental, and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems.

Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism.

In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5′ end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5′ end by the cognate factor. Enzymatic removal of 5′-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32–34 nt ribosome-protected fragments. Using this ‘in vivo ribosome footprinting’, we identified the function and molecular target of two candidate trans-acting factors.

Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa)

Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes.

A Two-Component Regulatory System in Transcriptional Control of Photosystem Stoichiometry: Redox-Dependent and Sodium Ion-Dependent Phosphoryl Transfer from Cyanobacterial Histidine Kinase Hik2 to Response Regulators Rre1 and RppA.

Two-component systems (TCSs) are ubiquitous signaling units found in prokaryotes. A TCS consists of a sensor histidine kinase and a response regulator protein as signal transducers. These regulatory systems mediate acclimation to various environmental changes by coupling environmental cues to gene expression. Hik2 is a sensor histidine kinase and its gene is found in all cyanobacteria. Hik2 is the homolog of Chloroplast Sensor Kinase (CSK), a protein involved in redox regulation of chloroplast gene expression during changes in light quality in plants and algae. Here we describe biochemical characterization of the signaling mechanism of Hik2 and its phosphotransferase activity. Results presented here indicate that Hik2 undergoes autophosphorylation on a conserved histidine residue, and becomes rapidly dephosphorylated by the action of response regulators Rre1 and RppA. We also show that the autophosphorylation of Hik2 is specifically inhibited by sodium ions.

Redox activity of thioredoxin z and fructokinase-like protein 1 is dispensable for autotrophic growth of Arabidopsis thaliana

Redox modulation of protein activity by thioredoxins (TRXs) plays a key role in cellular regulation. Thioredoxin z (TRX z) and its interaction partner fructokinase-like protein 1 (FLN1) represent subunits of the plastid-encoded RNA polymerase (PEP), suggesting a role of both proteins in redox regulation of chloroplast gene expression. Loss of TRX z or FLN1 expression generates a PEP-deficient phenotype and renders the plants incapable to grow autotrophically. This study shows that PEP function in trx z and fln1 plants can be restored by complementation with redox-inactive TRX z C106S and FLN1 C105/106A protein variants, respectively. The complemented plants showed wild-type levels of chloroplast gene expression and were restored in photosynthetic capacity, indicating that redox regulation of PEP through TRX z/FLN1 per se is not essential for autotrophic growth. Promoter–reporter gene studies indicate that TRX z and FLN1 are expressed during early phases of leaf development while expression ceases at maturation. Taken together, our data support a model in which TRX z and FLN1 are essential structural components of the PEP complex and their redox activity might only play a role in the fine tuning of PEP function.

Multiple Checkpoints for the Expression of the Chloroplast-Encoded Splicing Factor MatK

The chloroplast genome of land plants contains only a single gene for a splicing factor, Maturase K (MatK). To better understand the regulation of matK gene expression, we quantitatively investigated the expression of matK across tobacco (Nicotiana tabacum) development at the transcriptional, posttranscriptional, and protein levels. We observed striking discrepancies of MatK protein and matK messenger RNA levels in young tissue, suggestive of translational regulation or altered protein stability. We furthermore found increased matK messenger RNA stability in mature tissue, while other chloroplast RNAs tested showed little changes. Finally, we quantitatively measured MatK-intron interactions and found selective changes in the interaction of MatK with specific introns during plant development. This is evidence for a direct role of MatK in the regulation of chloroplast gene expression via splicing. We furthermore modeled a simplified matK gene expression network mathematically. The model reflects our experimental data and suggests future experimental perturbations to pinpoint regulatory checkpoints.

Transcriptional and posttranscriptional regulation of chloroplast gene expression by heavy metals in barley seedlings

The effects of Cd, Cu, and Ni ions on chloroplast gene expression were studied in 7-day-old barley (Hordeum vulgare L.) seedlings. Toxicity of heavy metals (HM) was estimated from their effects on root and shoot growth and from the accumulation of HM in roots and leaves of seedlings grown in roll culture. Root growth was affected by HM to a higher extent than shoot growth. A statistically significant inhibition of root growth was observed at concentrations of all studied HM ≥100 μM. Leaf growth was inhibited by Cu and Ni at concentrations ≥400 μM, while the inhibitory effect of Cd on leaf growth was observed at concentrations ≥100 μM. The translocation of Cu from roots to shoots of barley seedlings proceeded slower than the accumulation of Cu in roots. Unlike Cu, Ni was actively accumulated by shoots in addition to its strong absorption by roots. Using the run-on transcription and RT-PCR (reverse transcription-polymerase chain reaction), we showed that HM at a concentration of 100 μM regulated the expression of individual chloroplast genes. The presence of either Cd, Cu, or Ni ions in the plant growth solution enhanced transcription of rpl16, rpl23, and rpl12 genes encoding ribosomal proteins, and of ndhA encoding the subunit 1 of plastid NADH-plastoquinone oxidoreductase. Transcription of sixteen other studied plastid genes was not affected by HM. The activation of transcription of the studied genes was not always accompanied by an increase in the transcript copy number. Northern blot analysis showed that Cd impaired the splicing of rpl16, a chloroplast gene that contains an intron. Thus, this study proves that HM regulate not only the transcriptional activity of chloroplast genes but also the splicing of rpl16 transcripts.

Interaction of the pentatricopeptide-repeat protein DELAYED GREENING 1 with sigma factor SIG6 in the regulation of chloroplast gene expression in Arabidopsis cotyledons

The pentatricopeptide-repeat (PPR) protein DELAYED GREENING 1 (DG1) has been shown to be involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis. To gain insight into the mode of DG1 action, we used a yeast two-hybrid screening approach and identified a partner, SIG6, which is a chloroplast sigma factor responsible for the transcription of plastid-encoded RNA polymerase (PEP)-dependent chloroplast genes in cotyledons. Further analysis showed that the C-terminal region of DG1 and the N-terminal region of SIG6 are responsible for such interactions. High-level expression of a truncated C-terminal DG1 in wild-type Arabidopsis caused a dominant-negative phenotype. The sig6 dg1 double mutant displayed a more severe chlorotic phenotype, and the PEP-dependent chloroplast gene transcripts were greatly reduced compared with transcript levels in the single mutants. Overexpression of SIG6 rescued the chlorophyll deficiency in dg1 cotyledons but not in young leaves. In addition, increased SIG6 promoted PEP-dependent chloroplast gene transcript accumulation in the dg1 mutant background. These results suggest that the interaction of DG1 and SIG6 is functionally significant in the regulation of PEP-dependent chloroplast gene transcription in Arabidopsis cotyledons.

Regulation of chloroplast gene expression: light activated translation

Regulation of chloroplast gene expression: light activated translation

A new in vitro translation system for non‐radioactive assay from tobacco chloroplasts: effect of pre‐mRNA processing on translation in vitro

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.

Inducible Expression, Enzymatic Activity, and Origin of Higher Plant Homologues of Bacterial RelA/SpoT Stress Proteins in Nicotiana tabacum

All living cells possess adaptive responses to environmental stress that are essential to ensuring cell survival. For motile organisms, this can culminate in avoidance or attractile behavior, but for sessile organisms such as plants, stress adaptation is a process of success or failure within the confines of a given environment. Nearly all bacterial species possess a highly evolved system for stress adaptation, known as the "stringent response." This ancient and ubiquitous regulatory response is mediated by production of a second messenger of general stress, the nucleotide guanosine-3',5'-(bis)pyrophosphate (ppGpp), which mediates reprogramming of the global transcriptional output of the cell. Accumulation of ppGpp is stress-induced through the enzymatic activation of the well known bacterial ppGpp synthetases, RelA and SpoT. We have recently discovered homologues of bacterial relA/spoT genes in the model plant Nicotiana tabacum. We hypothesize that these homologues (designated RSH genes for RelA/SpoT homologues) serve a stress-adaptive function in plants analogous with their function in bacteria. In support of this hypothesis, we find 1) inducibility of tobacco RSH gene expression following treatment with jasmonic acid; 2) bona fide ppGpp synthesis activity of purified recombinant Nt-RSH2 protein, and 3) a wide spread distribution of RSH gene expression in the plant kingdom. Phylogenetic analyses identifies a distinct phylogenetic branch for the plant RSH proteins with two subgroups and supports ancient symbiosis and nuclear gene transfer as a possible origin for these stress response genes in plants. In addition, we find that Nt-RSH2 protein co-purifies with chloroplasts in subcellular fractionation experiments. Taken together, our findings implicate a direct mode of action of these ppGpp synthetases with regard to plant physiology, namely regulation of chloroplast gene expression in response to plant defense signals.

Involvement of G-proteins, calmodulin and tagetitoxin-sensitive RNA polymerase in light-regulated expression of plastid genes ( psbA, psaA and rbcL) in rice ( Oryza sativa L.)

The regulation of chloroplast gene expression by light involves multiple signaling components. In an earlier study, we demonstrated the role of calcium and phosphorylation in regulating the expression of photosynthesis-related plastid genes, psbA, psaA and rbcL, using rice as a model monocot system. This work has been extended further to examine the possible involvement of heterotrimeric GTP-binding proteins and calmodulin. Vacuum infiltration of 5-day-old etiolated rice seedlings with G-protein agonists, cholera toxin and GTPγS, increased the steady-state transcript levels of the plastid genes. The antagonists/inhibitors of calmodulin action, trifluoperazine and W7, inhibited the light-induced increase in steady-state transcript levels of these genes. The light-regulated expression of photosynthetic genes was also adversely affected by tagetitoxin, a specific inhibitor of plastid-encoded RNA polymerase. These results indicate the involvement of various signaling components in transduction of light signal that probably also recruits PEP to regulate plastid gene expression.

The stem-loop region of the tobacco psbA 5'UTR is an important determinant of mRNA stability and translation efficiency.

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs) of chloroplast transcripts. So far, only a few cis-acting elements within such 5'UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5'UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5'UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5'UTR stem-loop are required for mRNA stabilisation and translation. The 5'-terminal segment of the rbcL 5'UTR did not enhance the stability or translational activity of chimeric uidA mRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5'UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.

Translational regulations as specific traits of chloroplast gene expression

Studies of protein synthesis in the chloroplast compartment have revealed a unique combination of translational autoregulations and trans-regulations due to the delivery of a variety of nuclear factors that act post-transcriptionally. We show how these two characteristics concur to set the major step in the regulation of chloroplast gene expression at the translational level, leading to a surprisingly low sensitivity of chloroplast protein synthesis in response to extensive changes in plastome copy number and transcript concentration.

Regulation of Chloroplast Gene Expression is Affected in ali, a Novel Tobacco Albino Mutant

An albino mutant designated ali was isolated from an M 2population derived from a tobacco ( Nicotiana tabacum L. ‘BY-4’) plant which had been irradiated with a 14N-ion beam at an early stage of embryonic development. Leaf mesophyll cells of ali were devoid of developed chloroplasts, and no stacked thylakoid membranes could be detected in the plastids. The reduction of pigment and the arrest of chloroplast development in ali were associated with distinct alterations in gene expression. Expression of plastid-encoded genes for the photosynthesis system ( rbcL, psbA) was greatly reduced in ali, but at the transcript level plastid-encoded genes for 23S rDNA and 16S rDNA expression were only slightly reduced. Transcription of the nuclear-encoded Lhc and rbcS genes in the mutant was similar to that in the wild-type. There was no accumulation of Rubisco-L, Rubisco-S, and PSII-D1 polypeptides in the ali mutant. The number of chloroplasts per cell was similar in both ali and wild-type plants. These results suggest that ali is a novel albino mutant in which chloroplast gene expression is affected.

Some aspects of carboxyl-terminal processing of precursor D1 protein in photosystem II

The carboxyl-terminal processing of precursor D1 protein (pD1), which is an essential process in the functional integration of Mn-cluster in photosystem II, is a typical example of nuclear regulation of chloroplast gene expression at the level of post-translation in eukaryotic organisms. The nuclear-encoded enzyme (CtpA) involved in this process has been purified and its three-dimensional structure has been determined. This enzyme is classified into a novel type of serine protease, as also shown by our recent results on its inhibitor specificity and the site-directed mutational analysis (collaboration with Pakrasi?s group). On the other hand, approximately a hundred of deduced sequence data are now available on the substrate pD1 for a wide variety of organisms ranging from prokaryotic cyanobacteria to higher plants. Recently, unique sequences of pD1 for dinoflagellates have been provided. We have investigated this proteolytic process from both the enzyme and the substrate using systems of different integrities in order to understand the mechanism of functional expression of the water-splitting machinery in photosystem II. In this poster, we will discuss some aspects of enzyme-substrate interaction in the carboxyl-terminal processing of pD1 in thylakoidal lumen, and speculate about the evolution of nuclear regulation of this process.

34] Chloroplast p54 Endoribonuclease

Much of the regulation of chloroplast gene expression has been assigned to stability determinants at the level of RNA precursors, including interactions of sequence and/or structural elements with their cognate binding proteins. A number of these proteins are known to assemble at the 5′-untranslated region (UTR) of chloroplast mRNAs, protecting them from exonucleolytic degradation and forming ribonucleoprotein (RNP) complexes involved in translational control. One of the proteins known to be controlled by phosphorylation is a 3′-endoribonuclease, which was first found to specifically bind to a conserved U-rich sequence element (UUUAUCU) of chloroplast precursor transcripts. This protein, which purifies as a monomeric polypeptide of an apparent molecular size of 54 kDa, was subsequently termed p54. This chapter presents the purification and properties of the p54 endoribonuclease from mustard (Sinapis alba), which might turn out to be a key player in posttranscriptional regulation of chloroplast gene expression. As a first step toward the purification of the p54 endonuclease, chloroplast protein extracts from mustard (S. alba) are prepared. Although the procedure has been developed for mustard, it has been found suitable for other higher plant species as well without significant modifications.