Regulation Of Chloroplast Genes Research Articles

The melatonin receptor CAND2 is involved in the regulation of photosynthesis and chloroplast gene expression in Arabidopsis thaliana under photooxidative stress

Melatonin is a well-known bioactive molecule able to mitigate photooxidative damage caused by excess light. Here we have shown that mutant Arabidopsis lines with disrupted genes for melatonin putative receptor CAND2/PMTR1 and GPA1 encoding the α-subunit of heterotrimeric G-protein were partially insensitive to melatonin treatment under high light stress. They exhibited a higher degree of photodamage due to a significantly decreased photosynthetic activity and diminished expression of chloroplast and nuclear-encoded genes and the corresponding proteins. A possible mechanism for melatonin-dependent regulation of chloroplast genes is associated with a change in the activity of the genes for chloroplast RNA polymerases. We conclude that under high light stress, melatonin may act as a hormone-like signaling molecule via the CAND2/PMTR1-mediated signaling pathway.

Redox and light signals in the partitioning of photosynthetic electrons

The light‐driven electron transport reactions of photosynthesis convert the free energy of sunlight into usable chemical energy. Plants contain two primary electron transport pathways: the linear electron transport from water to NADP+, producing NADPH and ATP, and the cyclic electron flow around photosystem I, generating just ATP. Recent research from our laboratory reveals that the sensory and gene regulatory function of a Chloroplast Sensor Kinase (CSK) is central to the partitioning of electrons into the linear and cyclic pathways. A conserved iron‐sulfur cluster enables CSK to sense the redox state of the electron carrier plastoquinone. CSK thus places the expression of multiple chloroplast genes under the regulatory control of plastoquinone. Through the phytochrome photoreceptor, light provides a further fail‐safe input into chloroplast gene regulation. The combined action of redox and light signals brings about stoichiometric changes in electron transport complexes with important implications for the flow of photosynthetic electrons.Support or Funding InformationPurdue University, Showalter Trust, and AgSEEDThe CSK‐based genetic switch in chloroplastsFigure 1

The diurnal logic of the expression of the chloroplast genome in Chlamydomonas reinhardtii.

Chloroplasts are derived from cyanobacteria and have retained a bacterial-type genome and gene expression machinery. The chloroplast genome encodes many of the core components of the photosynthetic apparatus in the thylakoid membranes. To avoid photooxidative damage and production of harmful reactive oxygen species (ROS) by incompletely assembled thylakoid protein complexes, chloroplast gene expression must be tightly regulated and co-ordinated with gene expression in the nucleus. Little is known about the control of chloroplast gene expression at the genome-wide level in response to internal rhythms and external cues. To obtain a comprehensive picture of organelle transcript levels in the unicellular model alga Chlamydomonas reinhardtii in diurnal conditions, a qRT-PCR platform was developed and used to quantify 68 chloroplast, 21 mitochondrial as well as 71 nuclear transcripts in cells grown in highly controlled 12 h light/12 h dark cycles. Interestingly, in anticipation of dusk, chloroplast transcripts from genes involved in transcription reached peak levels first, followed by transcripts from genes involved in translation, and finally photosynthesis gene transcripts. This pattern matches perfectly the theoretical demands of a cell “waking up” from the night. A similar trend was observed in the nuclear transcripts. These results suggest a striking internal logic in the expression of the chloroplast genome and a previously unappreciated complexity in the regulation of chloroplast genes.

Expression of the Arabidopsis Sigma Factor SIG5 Is Photoreceptor and Photosynthesis Controlled.

Two collections of Arabidopsis GAL4 enhancer trap lines were screened for light-intensity dependent reporter gene activation. Line N9313 was isolated for its strong light-intensity regulation. The T-DNA element trapped distant enhancers of the SIG5 promoter, which drives expression of a sigma factor involved in regulation of chloroplast genes for photosystem II core proteins. The T-DNA insertion 715 bp upstream of the transcription initiation site splits the promoter in a distal and proximal part. Both parts are sensitive to blue and red light and depend on photosynthetic electron transport activity between photosystem II and the plastoquinone pool. The mainblue-light sensitivity is localized within a 196-bp sequence (–887 to –691 bp) in the proximal promoter region It is preferentially CRY1 and PHYB controlled. Type-I and type-II phytochromes mediate red-light sensitivity via various promoter elements spread over the proximal and distal upstream region. This work characterizes SIG5 as an anterograde control factor of chloroplast gene expression, which is controlled by chloroplast signals in a retrograde manner.

SIG1, a Sigma Factor for the Chloroplast RNA Polymerase, Differently Associates with Multiple DNA Regions in the Chloroplast Chromosomes in Vivo

Chloroplasts have their own DNA and gene expression systems. Transcription in chloroplasts is regulated by two types of RNA polymerase, nuclear-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP), and multiple sigma factors for PEP. To study transcriptional regulation in chloroplasts, a molecular genetic approach has extensively been used. However, this method may include indirect effects, and it cannot be applied to the analysis of factors essential to survival. These limitations make understanding specific regulation by transcription factors difficult. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool for obtaining information on transcription-factor binding sites; it can directly detect dynamic changes in their interaction patterns in vivo. To further understand transcriptional regulation in chloroplasts, we here established a ChIP-based method in Arabidopsis thaliana and analyzed the binding pattern of a chloroplast sigma factor, SIG1. We found that SIG1 specifically binds to newly identified target promoters as well as to a set of promoters of genes whose mRNA expression is dependent on OsSIG1 in rice and that this binding changed in response to high-light stress. These results suggested that the ChIP-based approach is very useful in understanding transcriptional regulation of chloroplast genes and can overcome several problems posed by conventional methods.

Motif analysis unveils the possible co-regulation of chloroplast genes and nuclear genes encoding chloroplast proteins.

Chloroplasts play critical roles in land plant cells. Despite their importance and the availability of at least 200 sequenced chloroplast genomes, the number of known DNA regulatory sequences in chloroplast genomes are limited. In this paper, we designed computational methods to systematically study putative DNA regulatory sequences in intergenic regions near chloroplast genes in seven plant species and in promoter sequences of nuclear genes in Arabidopsis and rice. We found that -35/-10 elements alone cannot explain the transcriptional regulation of chloroplast genes. We also concluded that there are unlikely motifs shared by intergenic sequences of most of chloroplast genes, indicating that these genes are regulated differently. Finally and surprisingly, we found five conserved motifs, each of which occurs in no more than six chloroplast intergenic sequences, are significantly shared by promoters of nuclear-genes encoding chloroplast proteins. By integrating information from gene function annotation, protein subcellular localization analyses, protein-protein interaction data, and gene expression data, we further showed support of the functionality of these conserved motifs. Our study implies the existence of unknown nuclear-encoded transcription factors that regulate both chloroplast genes and nuclear genes encoding chloroplast protein, which sheds light on the understanding of the transcriptional regulation of chloroplast genes.

Intracompartmental and Intercompartmental Transcriptional Networks Coordinate the Expression of Genes for Organellar Functions

Genes for mitochondrial and chloroplast proteins are distributed between the nuclear and organellar genomes. Organelle biogenesis and metabolism, therefore, require appropriate coordination of gene expression in the different compartments to ensure efficient synthesis of essential multiprotein complexes of mixed genetic origin. Whereas organelle-to-nucleus signaling influences nuclear gene expression at the transcriptional level, organellar gene expression (OGE) is thought to be primarily regulated posttranscriptionally. Here, we show that intracompartmental and intercompartmental transcriptional networks coordinate the expression of genes for organellar functions. Nearly 1,300 ATH1 microarray-based transcriptional profiles of nuclear and organellar genes for mitochondrial and chloroplast proteins in the model plant Arabidopsis (Arabidopsis thaliana) were analyzed. The activity of genes involved in organellar energy production (OEP) or OGE in each of the organelles and in the nucleus is highly coordinated. Intracompartmental networks that link the OEP and OGE gene sets serve to synchronize the expression of nucleus- and organelle-encoded proteins. At a higher regulatory level, coexpression of organellar and nuclear OEP/OGE genes typically modulates chloroplast functions but affects mitochondria only when chloroplast functions are perturbed. Under conditions that induce energy shortage, the intercompartmental coregulation of photosynthesis genes can even override intracompartmental networks. We conclude that dynamic intracompartmental and intercompartmental transcriptional networks for OEP and OGE genes adjust the activity of organelles in response to the cellular energy state and environmental stresses, and we identify candidate cis-elements involved in the transcriptional coregulation of nuclear genes. Regarding the transcriptional regulation of chloroplast genes, novel tentative target genes of σ factors are identified.

Oxidation–reduction signalling components in regulatory pathways of state transitions and photosystem stoichiometry adjustment in chloroplasts

State transitions and photosystem stoichiometry adjustment are two oxidation-reduction (redox)-regulated acclimatory responses in photosynthesis. State transitions are short-term adaptations that, in chloroplasts, involve reversible post-translational modification by phosphorylation of light-harvesting complex II (LHC II). Photosystem stoichiometry adjustments are long-term responses involving transcriptional regulation of reaction centre genes. Both responses are initiated by changes in light quality and are regulated by the redox state of plastoquinone (PQ). The LHC II kinase involved in the state 2 transition is a serine/threonine kinase known as STT7 in Chlamydomonas, and as STN7 in Arabidopsis. The phospho-LHC II phosphatase that produces the state 1 transition is a PP2C-type protein phosphatase currently termed both TAP38 and PPH1. In plants and algae, photosystem stoichiometry adjustment is governed by a modified two-component sensor kinase of cyanobacterial origin - chloroplast sensor kinase (CSK). CSK is a sensor of the PQ redox state. Chloroplast sigma factor 1 (SIG1) and plastid transcription kinase (PTK) are the functional partners of CSK in chloroplast gene regulation. We suggest a signalling pathway for photosystem stoichiometry adjustment. The signalling pathways of state transitions and photosystem stoichiometry adjustments are proposed to be distinct, with the two pathways sensing PQ redox state independently of each other.

Chloroplast biotechnology, genomics and evolution: current status, challenges and future directions

Chloroplast biotechnology, genomics and evolution: current status, challenges and future directions

Transients in chloroplast gene transcription

Transcriptional regulation of chloroplast genes is demonstrated by Quantitative Polymerase Chain Reaction (qPCR). These genes encode apoproteins of the reaction centres of photosystem I and photosystem II. Their transcription is regulated by changes in wavelength of light selectively absorbed by photosystem I and photosystem II, and therefore by the redox state of an electron carrier located between the two photosystems. Chloroplast transcriptional redox regulation is shown to have greater amplitude, and the kinetics of transcriptional changes are more complex, than suggested by previous experiments using only DNA probes in Northern blot experiments. Redox effects on chloroplast transcription appear to be superimposed on an endogenous rhythm of mRNA abundance. The functional significance of these transients in chloroplast gene transcription is discussed.

Differential expression of three plastidial sigma factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice.

We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative sigma-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.

Cooperation of Endo- and Exoribonucleases in Chloroplast mRNA Turnover

Chloroplasts were acquired by eukaryotic cells through endosymbiosis and have retained their own gene expression machinery. One hallmark of chloroplast gene regulation is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels. This review focuses on how chloroplast mRNA stability is regulated, through an examination of poly(A)-dependent and independent pathways. The poly(A)-dependent pathway is catalyzed by polynucleotide phosphorylase (PNPase), which both adds and degrades destabilizing poly(A) tails, whereas RNase II and PNPase may both participate in the poly(A)-independent pathway. Each system is initiated through endonucleolytic cleavages that remove 3' stem-loop structures, which are catalyzed by the related proteins CSP41a and CSP41b and possibly an RNase E-like enzyme. Overall, chloroplasts have retained the prokaryotic endonuclease-exonuclease RNA degradation system despite evolution in the number and character of the enzymes involved. This reflects the presence of the chloroplast within a eukaryotic host and the complex responses that occur to environmental and developmental cues.

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5' to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl2 and CaCl2. Although Mg2+- and Ca2+-activated CSP41 cleave at identical sites in the single-stranded regions of a stem-loop-containing substrate, Mg2+-activated CSP41 was also able to cleave within the double-stranded region of the stem-loop. Mixed metal experiments with Mg2+ and Ca2+ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn2+, Co2+ and Zn2+ compete with Mg2+ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg2+ (K(A,Mg) = 2.1 +/- 0.4 mM) is of the same order of magnitude as the stromal Mg2+ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg2+ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.

Light regulation of photosynthetic genes

Light influences plant development at various levels of gene expression. Preferentially photosynthetic genes are light controlled. In the nuclear‐cytosolic compartment this regulation occurs primarily at the level of transcription. To date, the light regulation of chloroplast genes has been observed mainly at the level of translation. However, mainly fully mature chloroplasts have been analyzed and the situation might be different in young chloroplasts, as has been found earlier for nuclear transcription. Finally, regulation also occurs by posttranslational modification. The involvement of the circadian clock in the regulation of gene expression is described in detail. During these studies it has been found that the circadian oscillator influences gene expression at various levels, probably resulting in the coordination of processes which might be harmful to the plant. The synthesis of chlorophylls as well as of their corresponding apoproteins are co‐regulated and reduce dangerous radical formation, especially at the higher light intensities. The maxima of chlorophyll and binding apoproteins occur during the first half of the light phase while other, presumably light‐insensitive processes, are shifted to the dark phase. Under light stress, e.g. full sunlight at 5°C, a different set of genes is activated; these include proteins such as catalases, peroxidases, superoxide dismutase or ELIPs, which are upregulated while the well known ‘light‐in‐ducible genes’ are down‐regulated under light stress conditions.

The psaA operon pre‐mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth

The chloroplast genome of Euglena gracilis contains a psaA operon which encodes a lysine tRNA gene, trnK; psaA and psaB photosystem I genes, and psbE, psbF, psbL and psbJ photosystem II genes. The pre-mRNA of the psaA operon undergoes a complex processing pathway of 5' and 3' tRNA processing, splicing of 11 group II introns and one group II twintron, plus three intercistronic RNA cleavage events. The accumulated transcripts of the psaA operon have been characterized by Northern hybridization, S1 nuclease analysis and primer extension RNA sequencing. The mature 5' end of the psaA-psaB-psbE-psbF-psbL-psbJ hexacistronic transcript lies 8 nt downstream of the trnK gene, and is the result of intercistronic trnK-psaA cleavage. Other intercistronic processing events occur between the psaA and psaB genes and the psaB and psbE genes. Processing at the latter site produces a dicistronic mRNA of PSI genes and a tetracistronic mRNA of PSII genes. The PSI dicistronic transcript is further processed to monocistronic psaA and psaB mRNAs. Secondary structural motifs within the intercistronic regions may be recognition sites for processing. The steady-state levels of psaA operon mRNAs from Euglena grown under several different conditions have been determined. Accumulated transcripts from all growth conditions are spliced, and a proportion are also processed at the intercistronic sites. The products of intercistronic processing increase from heterotrophic dark- to heterotrophic light-grown Euglena, and from heterotrophic light- to photoautotrophic light-grown Euglena. The differential accumulation of psaA operon mRNAs may be a means of chloroplast gene regulation or, alternatively, a consequence of gene expression during chloroplast development.

PHOTOSYNTHETIC INHIBITION IN STRAWBERRY GROWN UNDER TWICE AMBIENT CO2

`Midway' and `Raritan' strawberry cultivars were grown for six weeks under ambient (350) or twice ambient (700 μmol mol-1) CO2 Photosynthetic inhibition (40-50%) was observed in both cultivars as indicated by reductions (P < 0.001) in CO2 assimilation, carboxylation efficiency, stomatal conductance, and apparent quantum yield. Growth under high CO2 did not affect Rubisco activity in either cultivar, whereas Rubisco activation state (%) was reduced (72.9 to 67.6) in `Midway' and increased (70.8 to 83.3) in `Raritan' compared to ambient CO2 grown plants. Soluble and Rubisco protein decreased 10-15% in `Midway' and 40-45% in `Raritan' when grown under high CO2 Thus, `Raritan' reduced the amount of protein partitioned into photosynthetic enzymes under conditions of photosynthetic inhibition, whereas `Midway' experiences Rubisco deactivation. The reduction in Rubisco protein appears to be under the regulation of chloroplast genes.

Chloroplast gene regulation: interaction of the nuclear and chloroplast genomes in the expression of photosynthetic proteins

The photosynthetic, chlorophyll-containing chloroplast is one developmental stage of the plastid organelle. Plastids are found in all plant cell types and contain their own genetic material as well as all the machinery necessary for DNA, RNA and protein synthesis. They have diverse structures, functions and protein contents dependent upon the plastid type, which is determined to a large degree by the tissue (leaf versus root, etc) in which the plastid resides. For example, chromoplasts are carotenoidrich, non-photosynthetic plastids (yellow, red or orange), often associated with flowers or fruit. Plastids found in roots or tubers are termed amyioplasts. Plastids other than chloroplasts lack many of the proteins associated with photosynthetic membranes, and have messenger RNA (mENA) populations which are distinct from those of chloroplasts [ 1 ] However, because each of these plastids is able to convert to one of the other plastid types (Kirk and Tinley-Bassett, The Plastih Elsevier/North Holland Press, 1978; Mayfield and Huff, Plant P&id 1986, 81:30-351, expression of chloroplast genes must be considered in relation to gene expression in plastid types other than chloroplasts.

Heme regulates expression of phycobiliprotein photogenes in the unicellular rhodophyte, Cyanidium caldarium

Allophycocyanin and phycocyanin in the red alga (Cyanidium caldarium) are chloroplast-encoded, light-harvesting accessory pigments composed of alpha and beta subunit polypeptides (17-19 kDa) to which 1 or more residues of the heme-derived bile pigment chromophore phycocyanobilin are attached by cysteinyl thioether linkages (Offner, G.D., and Troxler, R.F. (1983) J. Biol. Chem. 258, 9931-9940). Western blot experiments utilizing phycobiliprotein antisera revealed that immunoreactive allophycocyanin and phycocyanin apoproteins were absent in cells grown in the dark and present in cells exposed to light. Northern blot experiments using genomic DNA hybridization probes indicated that phycobiliprotein mRNAs were absent in the dark, whereas cells exposed to light contained two allophycocyanin mRNA transcripts, 1.4 and 1.6 kilobases in length, and one phycocyanin mRNA transcript, 3.0 kilobases in length, providing evidence that phycobiliproteins are encoded in photogenes which are only transcriptionally active in the light. Northern and Western analyses demonstrated that cells incubated in the dark with the heme precursor delta-aminolevulinate contained allophycocyanin and phycocyanin mRNAs and apoproteins, indistinguishable in size, number, and quantity from those made in the light. Cells incubated in the dark with delta-aminolevulinate, protoporphyrin IX, or heme, but not biliverdin or phycocyanobilin, synthesized allophycocyanin and phycocyanin alpha and beta apoproteins, suggesting a role for heme in the control phycobiliprotein gene expression. Cells incubated with heme in the dark produced allophycocyanin and phycocyanin mRNA transcripts, but did not produce mRNAs for four other photogenes coding for a P-700 reaction center protein, a 32-kDa herbicide-binding protein, and the large and small subunits of ribulose-bisphosphate carboxylase. These results show, for the first time, that heme is a regulatory factor specifically involved in transcriptional regulation of chloroplast genes for phycobiliproteins.

Protein synthesis by isolated chloroplasts

Isolated chloroplasts show substantial rates of protein synthesis when illuminated. This 'in organello' protein synthesis system has been advantageously utilised to elucidate the coding capacity of chloroplast and the regulation of chloroplast genes. The system is also being used recently to transcribe and translate homologous and heterologous templates. In this mini-review, we attempt to critically ecaluate the available literature and present the current and the prospective lines of research.

Plastid run-on transcription. Application to determine the transcriptional regulation of spinach plastid genes.

We have developed a spinach plastid run-on transcription system to determine the extent of transcriptional regulation of chloroplast genes during morphogenetic changes of the organelle. In contrast to transcription in a spinach chloroplast extract, which requires initiation of exogenously added genes (Gruissem, W., Greenberg, B. M., Zurawski, G., and Hallick, R. B. (1983) Cell 35, 815-828), RNA synthesis in the run-on system is not affected by heparin or different salt concentrations. Transcription is asymmetric, and the size of the run-on transcripts varies between 75 nucleotides and 8 kilobases. Quantitative filter hybridization studies included gene-specific probes for the ribosomal RNA genes and nine protein-coding genes. Based on the amounts of hybridizable run-on transcripts, these genes can be ordered according to their respective transcriptional activities. The relative transcriptional activities of psbA, rbcL, and atpB in the run-on assay correlate closely with their reported promoter strengths in vitro. The plastid run-on transcription assay has been applied to determine the transcriptional regulation of plastid genes. Hybridization of run-on transcripts to regions of the spinach chloroplast genome containing at least nine tRNA genes indicates that most or all loci are highly transcribed. No significant qualitative and quantitative differences are detected when run-on transcripts from plastids of etiolated and greening cotyledons are hybridized to total, restriction enzyme-digested chloroplast DNA, demonstrating limited transcriptional regulation during chloroplast development.