Plastid Transcription Apparatus Research Articles

The role of PAP4/FSD3 and PAP9/FSD2 in heat stress responses of chloroplast genes

Chloroplasts’ mechanisms of adaptation to elevated temperatures are largely determined by the gene expression of the plastid transcription apparatus. Gene disruption of iron-containing superoxide dismutase PAP4/FSD3 and PAP9/FSD2, which are parts of the DNA–RNA polymerase complex of plastids, contributed to a decrease in resistance to oxidative stress caused by the prolonged action of elevated temperatures (5 days, 30 °C). Under heat stress conditions, pap4/fsd3 and pap9/fsd2 mutants showed a decline in chlorophyll content and photosynthesis level, as measured by photosynthetic parameters, and a different amplitude of HSP gene response to heat stress. The expression of nuclear- and plastid-encoded photosynthesis genes and corresponding proteins was strongly inhibited in the mutants as compared with wild-type plants and was further suppressed or displayed no additional changes at 30 °C. NEP-dependent plastid genes, as well as NEP genes RPOTp and RPOTmp, were also downregulated in the mutants by high temperature or remained stable, unlike in wild-type seedlings where these genes were strongly upregulated. The results obtained correspond to the concept of the complex effect of various forms of reactive oxygen species under all types of stresses, including heat stress, and confirm the hypothesis of a new regulatory function in plastid transcription acquired by enzymatic proteins during evolution.

Plastid Gene Transcription: An Update on Promoters and RNA Polymerases.

Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and leucoplasts), they form a group of intracellular biosynthetic machines collectively known as plastids. These plant cell constituents have their own genome (plastome), their own (70S) ribosomes, and complete enzymatic equipment covering the full range from DNA replication via transcription and RNA processive modification to translation. Plastid RNA synthesis (gene transcription) involves the collaborative activity of two distinct types of RNA polymerases that differ in their phylogenetic origin as well as their architecture and mode of function. The existence of multiple plastid RNA polymerases is reflected by distinctive sets of regulatory DNA elements and protein factors. This complexity of the plastid transcription apparatus thus provides ample room for regulatory effects at many levels within and beyond transcription. Research in this field offers insight into the various ways in which plastid genes, both singly and groupwise, can be regulated according to the needs of the entire cell. Furthermore, it opens up strategies that allow to alter these processes in order to optimize the expression of desired gene products.

Cytokinins and Abscisic Acid Regulate the Expression of the Genes for Plastid Transcription Apparatus during Heat Shock

The treatment of Arabidopsis thaliana plants with exogenous cytokinin (CK) followed by heat shock (HS) activated the expression of the genes for the plastid transcription machinery but adversely affected the plant viability. Abscisic acid (ABA), conversely, promoted maintaining the resistance to HS and had differentially affected different components of the plastid transcriptional complex. This hormone suppressed the accumulation of transcripts of PEP genes and the genes encoding PAP proteins, which are involved in DNA-RNA metabolism. However, it had no effect or activated the expression of NEP genes and PAP genes, which are involved in the redox regulation, as well as the genes encoding the stress-inducible trans-factor (SIG5) and the plastid transcription Ser/Thr protein kinase (spCK2). Thus, for the adaptation of plants to elevated temperatures, both increase and decrease in the expression of the genes for the plastid transcriptional machinery with the involvement of various regulatory systems, including phytohormones, are equally significant.

Cytokinins and Abscisic Acid Regulate the Expression of the Genes for Plastid Transcription Apparatus during Heat Shock.

The treatment of Arabidopsis thaliana plants with exogenous cytokinin (CK) followed by heat shock (HS) activated the expression of the genes for the plastid transcription machinery but adversely affected the plant viability. Abscisic acid (ABA), conversely, promoted maintaining the resistance to HS and had differentially affected different components of the plastid transcriptional complex. This hormone suppressed the accumulation of transcripts of PEP genes and the genes encoding PAP proteins, which are involved in DNA-RNA metabolism. However, it had no effect or activated the expression of NEP genes and PAP genes, which are involved in the redox regulation, as well as the genes encoding the stress-inducible trans-factor (SIG5) and the plastid transcription Ser/Thr protein kinase (cpCK2). Thus, for the adaptation of plants to elevated temperatures, both increase and decrease in the expression of the genes for the plastid transcriptional machinery with the involvement of various regulatory systems, including phytohormones, are equally significant.

Phytohormones Regulate the Expression of Nuclear Genes Encoding the Components of the Plastid Transcription Apparatus.

As multifunctional regulators of physiological processes, phytohormones play an important role in the regulation of expression of the plastid genome and chloroplast biogenesis. Hormones can directly regulate the expression of genes localized in the chloroplast genome. However, many components of the plastid transcription apparatus are encoded by nuclear rather than plastid genes. It remains obscure whether these nuclear genes are subject to hormonal regulation. This is the first study to show that phytohormones exert differential effects on the expression of nuclear genes of the transcription machinery of the Arabidopsis thaliana plastome. RT-PCR analysis showed that the level of transcripts of the majority of studied genes was activated by trans-zeatin but decreased under the influence of ABA, methyl jasmonate, and salicylic acid, whereas ethylene had no significant effect, and the effects of brassinolide depended on the illumination conditions. The results of this study indicate that the hormonal regulation of the plastome expression can be mediated by differential regulation of the nuclear genes encoding plastid transcription machinery components.

Cytokinin membrane receptors modulate transcript accumulation of plastid encoded genes

Cytokinins (CKs) have a strong influence on plastid development. We used the Arabidopsis thaliana (L.) Heynh. loss-of function insertion mutants ahk2, ahk3 and ahk4 alone or in different combinations to examine the effect of individual membrane cytokinin (CK) receptors on the expression of plastid genes and nuclear genes for the plastid transcription machinery. Quantitative real-time PCR-analyses demonstrate that the CK membrane receptors have overlapping functions in regulating the levels of plastid house-keeping genes in 7-day-old seedlings, relative to the nuclear UBQ10 gene. In contrast, photosynthesis genes responded differently to AHK3 and AHK2 receptor inactivation and the results show a dominant role of the AHK2/AHK3 receptor combination in the CK responses. These differences could be, at least in part, mediated through the regulation by the nuclear-encoded RPOTp and RPOTmp genes for plastid phage-type RNA polymerases. CK positively regulated the transcript abundance of the nuclear encoded SIG2 and SIG6, and negatively that of SIG4 and SIG5 for sigma factors of the plastid transcription apparatus. We propose that these two sigma factors may affect the CK-controlled plastid gene expression.

Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation

Arabidopsis seed formation is coupled with two plastid differentiation processes. Chloroplast formation starts during embryogenesis and ends with the maturation phase. It is followed by chloroplast dedifferentiation/degeneration that starts at the end of the maturation phase and leads to the presence of small non-photosynthetic plastids in dry seeds. We have analysed mRNA and protein levels of nucleus- and plastid-encoded (NEP and PEP) components of the plastid transcriptional machinery, mRNA and protein levels of some plastid RNA polymerase target genes, changes in plastid transcriptome profiles and mRNA and protein levels of some selected nucleus-encoded plastid-related genes in developing seeds during embryogenesis, maturation and desiccation. As expected, most of the mRNAs and proteins increase in abundance during maturation and decrease during desiccation, when plastids dedifferentiate/degenerate. In contrast, mRNAs and proteins of components of the plastid transcriptional apparatus do not decrease or even still increase during the period of plastid dedifferentiation. Results suggest that proteins of the plastid transcriptional machinery are specifically protected from degradation during the desiccation period and conserved in dry seeds to allow immediate regain of plastid transcriptional activity during stratification/germination. In addition, results reveal accumulation and storage of mRNAs coding for RNA polymerase components and sigma factors in dry seeds. They should provide immediately-to-use templates for translation on cytoplasmic ribosomes in order to enhance RNA polymerase protein levels and to provide regulatory proteins for stored PEP to guaranty efficient plastid genome transcription during germination.

Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?

Plastid gene expression is rather complex. Transcription is performed by three different RNA polymerases, two of them are nucleus-encoded, monomeric, of the phage-type (named RPOTp and RPOTmp) and one of them is plastid-encoded, multimeric, of the eubacterial-type (named PEP). The activity of the eubacterial-type RNA polymerase is regulated by up to six nucleus-encoded transcription initiation factors of the sigma-type. This complexity of the plastid transcriptional apparatus is not yet well understood and raises the question of whether it is subject to any regulation or just ensures constitutive transcription of the plastid genome. On the other hand, considerable advances have been made during the last years elucidating the role of sigma factors for specific promoter recognition and selected transcription of some plastid genes. Sigma-interacting proteins have been identified and phosphorylation-dependent functional changes of sigma factors have been revealed. The present review aims to summarize these recent advances and to convince the reader that plastid gene expression is regulated on the transcriptional level by sigma factor action.

Role and regulation of plastid sigma factors and their functional interactors during chloroplast transcription – Recent lessons from Arabidopsis thaliana

Chloroplasts and other plastids within plant cells together are responsible for autotrophic growth and biosynthesis of metabolic key components. Genetically, the plastids are hybrid organelles composed of proteins that are either products of their own organellar genes or are nucleus-encoded and imported from the cytosol. This dual genetic principle is evident in the case of the multi-subunit RNA polymerase, i.e. a major enzyme of the plastid transcription apparatus, consisting of an organelle-encoded core surrounded by imported regulatory proteins. Representatives of the latter are the members of the plastid sigma factor family as well as a Ser/Thr-protein kinase (PTK/cpCK2) that functionally modifies these factors and controls transcription. The plant sigma factors contain regulatory phosphoacceptor sites within their unconserved (factor-specific) portion that precedes the conserved catalytic region. Phosphorylation state changes of these regulatory sites help establish the activity and promoter selectivity of individual members of this plant transcription factor family. The protein kinase itself responds to SH-group regulation by glutathione and transmits the redox signal via its phosphorylation activity to the plastid transcription apparatus. Other functional interactors include a set of sigma binding proteins that confer enhanced promoter binding in vitro and are thought to be involved in pathogenic stress responses of the chloroplast in vivo.

Specific function of a plastid sigma factor for ndhF gene transcription

The complexity of the plastid transcriptional apparatus (two or three different RNA polymerases and numerous regulatory proteins) makes it very difficult to attribute specific function(s) to its individual components. We have characterized an Arabidopsis T-DNA insertion line disrupting the nuclear gene coding for one of the six plastid sigma factors (SIG4) that regulate the activity of the plastid-encoded RNA polymerase PEP. This mutant shows a specific diminution of transcription of the plastid ndhF gene, coding for a subunit of the plastid NDH [NAD(P)H dehydrogenase] complex. The absence of another NDH subunit, i.e. NDHH, and the absence of a chlorophyll fluorescence transient previously attributed to the activity of the plastid NDH complex indicate a strong down-regulation of NDH activity in the mutant plants. Results suggest that plastid NDH activity is regulated on the transcriptional level by an ndhF-specific plastid sigma factor, SIG4.

The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation.

The rice virescent-2 mutant (v(2)) is temperature conditional and develops chlorotic, chloroplast-deficient leaves at the restrictive temperature. In the v(2) mutant, plastid-encoded proteins involved in photosynthesis and plastid transcriptional regulation were not detectable at any time during chloroplast differentiation. However, the plastid transcripts for these two classes of proteins behaved differently in the mutant, with those for the plastid transcription/translation apparatus accumulating to wild-type levels and those for photosynthetic apparatus being suppressed. Polysome analysis showed that translation of the plastid transcripts encoding the plastid transcription/translation apparatus was blocked at an early stage of chloroplast differentiation. Accumulation of transcripts of nuclear-encoded photosynthetic genes, such as cab and rbcS, was strongly suppressed in the mutant at later stages of chloroplast differentiation, whereas transcripts of genes for the plastid transcription apparatus, such as OsRpoTp and OsSIG2A, accumulated to abnormally high levels at these stages. These results suggest that activation of the plastid translation machinery at an early stage of chloroplast differentiation is important for triggering the transmission of information about plastid developmental state to the nucleus, which in turn is required for the induction of nuclear-encoded chloroplast proteins at later stages of chloroplast differentiation.

The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS.

Etched1 (et1) is a pleiotropic, recessive mutation of maize that causes fissured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wild-type plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was significantly reduced in chloroplasts of et1-R plants. ET1 is the first zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.

The plastid transcription kinase from mustard (Sinapis albaL.)

The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the alpha subunit of cytosolic casein kinase (CK2) that contained an N-terminal extension for a putative plastid transit peptide. Using in organello chloroplast import studies, immunodetection and MS, we found that the corresponding protein, termed cpCK2alpha, is targeted to the chloroplast and is associated with the plastid RNA polymerase PEP-A. The bacterially overexpressed protein shows CK2 kinase activity and is subject to glutathione inhibition in the same way as authentic chloroplast PTK. Furthermore, it readily phosphorylates components of the plastid transcription apparatus in vitro with a substrate specificity similar to that of PTK.

Green life: Control of chloroplast gene transcription

AbstractChloroplasts and other plastids are plant cell organelles that account for major biochemical functions. They contain their own gene expression system but are integrated into the signaling network of the entire cell. Both nuclear and plastid genes are involved in chloroplast biogenesis, and the gene expression pathways both inside and outside the organelle are subject to developmental and environmental control. The plastid transcription apparatus reflects this general scheme, with a basic organelle‐encoded enzymatic machinery surrounded by factors that may be encoded by nuclear genes. Among the transcription regulatory mechanisms thought to play a role during plastid development are: (1) differential usage of promoter elements; (2) phosphorylation of transcription factors by a protein kinase, which is itself subject to phosphorylation and redox control; (3) dynamic changes in the composition of the transcription apparatus. In etioplasts, the dominating polymerase ‘B’ is a bacterial‐type enzyme, whereas the major chloroplast polymerase ‘A’ is a much larger enzyme reminiscent of those in the nucleus. These two enzyme forms may share common components and recruit others during development.

Changes in RNA-Polymerase Activity during Biogenesis, Maturation and Senescence of Barley Chloroplasts. Comparative Analysis of Transcripts Synthesized Either in Run-on Assays or by Transcriptionally Active Chromosomes

Summary The relative transcription rates of plastid genes were analyzed by hybridization of both run-on transcripts and TAC (transcriptionally active chromosome) derived transcripts to filter bound barley plastid DNA fragments. The results clearly demonstrate that TACs are able to transcribe all classes of genes. Development related changes in transcription were characterized by hybridization to 16 specific barley plastid DNA fragments of the transcripts derived from proplastids and from four physiologically well characterized stages of chloroplast biogenesis and senescence. Both in vitro transcription with TAC and run-on assays revealed a differential transcription of plastid genes. During biogenesis and maturation of chloroplasts relative transcription of rDNA decreases while relative transcription of psbA , psbD and psaB increases in parallel. Senescence is accompanied by converse changes in transcription. Though the main development related changes in plastid DNA transcription observed in run-on assays are retained by TAC, transcription of some genes ( atpA , rbcL , rpl , rps , rpoB ) is less pronounced in TAC preparations than in run-on assays. Differences between the two transcription systems are particularly obvious in the case of photosynthetically competent chloroplasts and might indicate that parts of the plastid transcriptional apparatus are not represented by TAC.