Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants.

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants. Previous studies have established that tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) play essential roles in plastid retrograde signaling during early chloroplast biogenesis; however, their functional relationship remains unknown. In this study, we generated a series of rice TPB-related gun (genome uncoupled) mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon (NF; a noncompetitive inhibitor of carotenoid biosynthesis) and/or lincomycin (Lin; a specific inhibitor of plastid translation). We show that under NF treatment, expression of plastid-encoded polymerase (PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants. We further demonstrate that the derepressed expression of PEP-transcribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome. In addition, we show that expression of photosynthesis-associated nuclear genes (PhANGs) and PEP-transcribed genes is correlated in the rice TPB-related gun mutants, with or without NF or Lin treatment. A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants. Moreover, we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants. Further, we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling. Taken together, our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

Arabidopsis Mitochondrial Transcription Termination Factor mTERF2 Promotes Splicing of Group IIB Introns.

Plastid gene expression (PGE) is essential for chloroplast biogenesis and function and, hence, for plant development. However, many aspects of PGE remain obscure due to the complexity of the process. A hallmark of nuclear-organellar coordination of gene expression is the emergence of nucleus-encoded protein families, including nucleic-acid binding proteins, during the evolution of the green plant lineage. One of these is the mitochondrial transcription termination factor (mTERF) family, the members of which regulate various steps in gene expression in chloroplasts and/or mitochondria. Here, we describe the molecular function of the chloroplast-localized mTERF2 in Arabidopsis thaliana. The complete loss of mTERF2 function results in embryo lethality, whereas directed, microRNA (amiR)-mediated knockdown of MTERF2 is associated with perturbed plant development and reduced chlorophyll content. Moreover, photosynthesis is impaired in amiR-mterf2 plants, as indicated by reduced levels of photosystem subunits, although the levels of the corresponding messenger RNAs are not affected. RNA immunoprecipitation followed by RNA sequencing (RIP-Seq) experiments, combined with whole-genome RNA-Seq, RNA gel-blot, and quantitative RT-PCR analyses, revealed that mTERF2 is required for the splicing of the group IIB introns of ycf3 (intron 1) and rps12.

Transcription initiation as a control point in plastid gene expression.

The extensive processing and protein-assisted stabilization of transcripts have been taken as evidence for a viewpoint that the control of gene expression had shifted entirely in evolution from transcriptional in the bacterial endosymbiont to posttranscriptional in the plastid. This suggestion is however at odds with many observations on plastid gene transcription. Chloroplasts of flowering plants and mosses contain two or more RNA polymerases with distinct promoter preference and division of labor for the coordinated synthesis of plastid RNAs. Plant and algal plastids further possess multiple nonredundant sigma factors that function as transcription initiation factors. The controlled accumulation of plastid sigma factors and modification of their activity by sigma-binding proteins and phosphorylation constitute additional transcriptional regulatory strategies. Plant and algal plastids also contain dedicated one- or two-component transcriptional regulators. Transcription initiation thus continues to form a critical control point at which varied developmental and environmental signals intersect with plastid gene expression.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

Arabidopsis Plastid-RNA Polymerase RPOTp Is Involved in Abiotic Stress Tolerance.

Plastid gene expression (PGE) must adequately respond to changes in both development and environmental cues. The transcriptional machinery of plastids in land plants is far more complex than that of prokaryotes. Two types of DNA-dependent RNA polymerases transcribe the plastid genome: a multimeric plastid-encoded polymerase (PEP), and a monomeric nuclear-encoded polymerase (NEP). A single NEP in monocots (RPOTp, RNA polymerase of the T3/T7 phage-type) and two NEPs in dicots (plastid-targeted RPOTp, and plastid- and mitochondrial-targeted RPOTmp) have been hitherto identified. To unravel the role of PGE in plant responses to abiotic stress, we investigated if Arabidopsis RPOTp could function in plant salt tolerance. To this end, we studied the sensitivity of T-DNA mutants scabra3-2 (sca3-2) and sca3-3, defective in the RPOTp gene, to salinity, osmotic stress and the phytohormone abscisic acid (ABA) required for plants to adapt to abiotic stress. sca3 mutants were hypersensitive to NaCl, mannitol and ABA during germination and seedling establishment. Later in development, sca3 plants displayed reduced sensitivity to salt stress. A gene ontology (GO) analysis of the nuclear genes differentially expressed in the sca3-2 mutant (301) revealed that many significantly enriched GO terms were related to chloroplast function, and also to the response to several abiotic stresses. By quantitative RT-PCR (qRT-PCR), we found that genes LHCB1 (LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING1) and AOX1A (ALTERNATIVE OXIDASE 1A) were respectively down- and up-regulated in the Columbia-0 (Col-0) salt-stressed plants, which suggests the activation of plastid and mitochondria-to-nucleus retrograde signaling. The transcript levels of genes RPOTp, RPOTmp and RPOTm significantly increased in these salt-stressed seedlings, but this enhanced expression did not lead to the up-regulation of the plastid genes solely transcribed by NEP. Similar to salinity, carotenoid inhibitor norflurazon (NF) also enhanced the RPOTp transcript levels in Col-0 seedlings. This shows that besides salinity, inhibition of chloroplast biogenesis also induces RPOTp expression. Unlike salt and NF, the NEP genes were significantly down-regulated in the Col-0 seedlings grown in ABA-supplemented media. Together, our findings demonstrate that RPOTp functions in abiotic stress tolerance, and RPOTp is likely regulated positively by plastid-to-nucleus retrograde signaling, which is triggered when chloroplast functionality is perturbed by environmental stresses, e.g., salinity or NF. This suggests the existence of a compensatory mechanism, elicited by impaired chloroplast function. To our knowledge, this is the first study to suggest the role of a nuclear-encoded plastid-RNA polymerase in salt stress tolerance in plants.

Determination of the DNA/RNA-Associated Subproteome from Chloroplasts and Other Plastid Types.

Plastids of plant and algae cells are of endosymbiotic origin. They possess their own genome and a sophisticated protein machinery to express it. Studies over the recent years uncovered that the regulation of plastid gene expression is highly complex involving a multiplicity of regulatory protein factors that are mostly imported from the cytosol. Proper expression of the chloroplast genome in coordination with nuclear genome was found to be absolutely essential for efficient growth and development of plants especially during early steps of photomorphogenesis, but also at later stages of the plant life cycle. Protein factors being responsible for such essential steps, therefore, are highly interesting for fundamental science as well as for industrial applications targeting crop improvement and yield increase. Nevertheless, many proteins involved in regulation of plastid gene expression are still unidentified and/or uncharacterized. This asks for appropriate methods to analyze this special subproteome. Here, we describe suitable methods that proved to be successful in the analysis of the plastid subproteome of DNA/RNA-binding proteins.

Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development.

In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem), cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

pTAC10, an S1-domain-containing component of the transcriptionally active chromosome complex, is essential for plastid gene expression in Arabidopsis thaliana and is phosphorylated by chloroplast-targeted casein kinase II.

In higher plant chloroplasts, the plastid-encoded RNA polymerase (PEP) consists of four catalytic subunits and numerous nuclear-encoded accessory proteins, including pTAC10, an S1-domain-containing protein. In this study, pTAC10 knockout lines were characterized. Two ptac10 mutants had an albino phenotype and severely impaired chloroplast development. The pTAC10 genomic sequence fused to a four-tandem MYC tag driven by its own promoter functionally complemented the ptac10-1 mutant phenotype. pTAC10 was present in both the chloroplast stroma and thylakoids. Two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE), and immunoblotting assays showed that pTAC10:MYC co-migrates with one of the PEP core subunits, RpoB. A comprehensive investigation of the plastid gene expression profiles by quantitative RT-PCR revealed that, compared with wild-type plants, the abundance of PEP-dependent plastid transcripts is severely decreased in the ptac10-1 mutant, while the amount of plastid transcripts exclusively transcribed by NEP either barely changes or even increases. RNA blot analysis confirmed that PEP-dependent chloroplast transcripts, including psaB, psbA and rbcL, substantially decrease in the ptac10-1 mutant. Immunoblotting showed reduced accumulation of most chloroplast proteins in the ptac10 mutants. These data indicate the essential role of pTAC10 in plastid gene expression and plastid development. pTAC10 interacts with chloroplast-targeted casein kinase 2 (cpCK2) in vitro and in vivo and can be phosphorylated by Arabidopsis cpCK2 in vitro at sites Ser95, Ser396 and Ser434. RNA-EMSA assays showed that pTAC10 is able to bind to the psbA, atpE and accD transcripts, suggesting a non-specific RNA-binding activity of pTAC10. The RNA affinity of pTAC10 was enhanced by phosphorylation and decreased by the amino acid substitution Ser434-Ala of pTAC10. These data show that pTAC10 is essential for plastid gene expression in Arabidopsis and that it can be phosphorylated by cpCK2.

Whole plastid transcriptomes reveal abundant RNA editing sites and differential editing status in Phalaenopsis aphrodite subsp. formosana.

BACKGROUND: RNA editing is a process of post-transcriptional level of gene regulation by nucleotide modification. Previously, the chloroplast DNA of Taiwan endemic moth orchid, P. aphrodite subsp. formosana was determined, and 44 RNA editing sites were identified from 24 plastid protein-coding transcripts of leaf tissue via RT-PCR and then conventional Sanger sequencing. However, the RNA editing status of whole-plastid transcripts in leaf and other distinct tissue types in moth orchids has not been addressed. To sensitively and extensively examine the plastid RNA editing status of moth orchid, RNA-Seq was used to investigate the editing status of whole-plastid transcripts from leaf and floral tissues by mapping the sequence reads to the corresponding cpDNA template. With the threshold of at least 5% C-to-U or U-to-C conversion events observed in sequence reads considered as RNA editing sites. RESULTS: In total, 137 edits with 126 C-to-U and 11 U-to-C conversions, including 93 newly discovered edits, were identified in plastid transcripts, representing an average of 0.09% of the nucleotides examined in moth orchid. Overall, 110 and 106 edits were present in leaf and floral tissues, respectively, with 79 edits in common. As well, 79 edits were involved in protein-coding transcripts, and the 58 nucleotide conversions caused the non-synonymous substitution. At least 32 edits showed significant (≧20%) differential editing between leaf and floral tissues. Finally, RNA editing in trnM is required for the formation of a standard clover-leaf structure. CONCLUSIONS: We identified 137 edits in plastid transcripts of moth orchid, the highest number reported so far in monocots. The consequence of RNA editing in protein-coding transcripts mainly cause the amino acid change and tend to increase the hydrophobicity as well as conservation among plant phylogeny. RNA editing occurred in non-protein-coding transcripts such as tRNA, introns and untranslated regulatory regions could affect the formation and stability of secondary structure, which might play an important role in the regulation of gene expression. Furthermore, some unidentified tissue-specific factors might be required for regulating RNA editing in moth orchid.

DELAYED GREENING 238, a Nuclear-Encoded Chloroplast Nucleoid Protein, Is Involved in the Regulation of Early Chloroplast Development and Plastid Gene Expression in Arabidopsis thaliana.

Chloroplast development is an essential process for plant growth that is regulated by numerous proteins. Plastid-encoded plastid RNA polymerase (PEP) is a large complex that regulates plastid gene transcription and chloroplast development. However, many proteins in this complex remain to be identified. Here, through large-scale screening of Arabidopsis mutants by Chl fluorescence imaging, we identified a novel protein, DELAYED GREENING 238 (DG238), which is involved in regulating chloroplast development and plastid gene expression. Loss of DG238 retards plant growth, delays young leaf greening, affects chloroplast development and lowers photosynthetic efficiency. Moreover, blue-native PAGE (BN-PAGE) and Western blot analysis indicated that PSII and PSI protein levels are reduced in dg238 mutants. DG238 is mainly expressed in young tissues and is regulated by light signals. Subcellular localization analysis showed that DG238 is a nuclear-encoded chloroplast nucleoid protein. More interestingly, DG238 was co-expressed with FLN1, which encodes an essential subunit of the PEP complex. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays showed that DG238 can also interact with FLN1. Taken together, these results suggest that DG238 may function as a component of the PEP complex that is important for the early stage of chloroplast development and helps regulate PEP-dependent plastid gene expression.

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.

Conditional repression of essential chloroplast genes: Evidence for new plastid signaling pathways.

The development of a repressible chloroplast gene expression system in Chlamydomonas reinhardtii has opened the door for studying the role of essential chloroplast genes. This approach has been used to analyze three chloroplast genes of this sort coding for the α subunit of RNA polymerase (rpoA), a ribosomal protein (rps12) and the catalytic subunit of the ATP-dependent ClpP protease (clpP1). Depletion of the three corresponding proteins leads to growth arrest and cell death. Shutdown of chloroplast transcription and translation increases the abundance of a set of plastid transcripts that includes mainly those involved in transcription, translation and proteolysis and reveals multiple regulatory feedback loops in the chloroplast gene circuitry. Depletion of ClpP profoundly affects plastid protein homeostasis and elicits an autophagy-like response with extensive cytoplasmic vacuolization of cells. It also triggers changes in chloroplast and nuclear gene expression resulting in increased abundance of chaperones, proteases, ubiquitin-related proteins and proteins involved in lipid trafficking and thylakoid biogenesis. These features are hallmarks of an unfolded protein response in the chloroplast and raise new questions on plastid protein homeostasis and plastid signaling. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Phytochrome-dependent coordinate control of distinct aspects of nuclear and plastid gene expression during anterograde signaling and photomorphogenesis.

Light perception by photoreceptors impacts plastid transcription, development, and differentiation. This photoreceptor-dependent activity suggests a mechanism for photoregulation of gene expression in the nucleus and plastid that serves to coordinate expression of critical genes of these two organelles. This coordinate expression is required for proper stoichiometric accumulation of components needed for assembly of plastids, photosynthetic light-harvesting complexes and components such as phytochromes. Chloroplast-targeted sigma factors, which function together with the plastid-encoded RNA polymerase to regulate expression of plastid-encoded genes, and nuclear-encoded plastid development factors, such as GLK1 and GLK2, are targets of phytochrome regulation. Such phytochrome-dependent functions are hypothesized to allow light-dependent regulation, and feasibly tuning, of plastid components and function in response to changes in the external environment, which directly affects photosynthesis and the potential for light-induced damage. When the size and protein composition of the light-harvesting complexes are not tuned to the external environment, imbalances in electron transport can impact the cellular redox state and cause cellular damage. We show that phytochromes specifically regulate the expression of multiple factors that function to modulate plastid transcription and, thus, provide a paradigm for coordinate expression of the nuclear and plastid genomes in response to changes in external light conditions. As phytochromes respond to changes in the prevalent wavelengths of light and light intensity, we propose that specific phytochrome-dependent molecular mechanisms are used during light-dependent signaling between the nucleus and chloroplast during photomorphogenesis to coordinate chloroplast development with plant developmental stage and the external environment.

PRDA1, a novel chloroplast nucleoid protein, is required for early chloroplast development and is involved in the regulation of plastid gene expression in Arabidopsis.

Chloroplast development requires accurate spatio-temporal expression of plastid genes. The regulation of plastid genes mediated by plastid-encoded RNA polymerase (PEP) is rather complex, and its related mechanism remains largely unclear. Here, we report the identification of a novel protein that is essential for plant development, PEP-Related Development Arrested 1 (PRDA1). Knock-out of PRDA1 in Arabidopsis (prda1 mutant) caused a seedling-lethal, albino phenotype and arrested the development of leaf chloroplasts. Localization analysis showed that PRDA1 was specifically targeted to chloroplasts and co-localized with chloroplast nucleoids, revealing that PRDA1 is a chloroplast nucleoid-associated protein. Gene expression analyses revealed that the PEP-dependent plastid transcript levels were greatly reduced in prda1. PRDA1 was co-expressed with most of the PEP-associated proteins. Protein interaction assays showed that PRDA1 clearly interacts with MRL7 and FSD2, both of which have been verified as essential for PEP-related chloroplast development. Reactive oxygen species scavenging through dimethylthiourea markedly alleviated the cotyledon-albino phenotypes of PRDA1 and MRL7 RNA interference seedlings. These results demonstrate that PRDA1 is required for early chloroplast development and involved in the regulation of plastid gene expression.

Phytochrome-induced SIG2 expression contributes to photoregulation of phytochrome signalling and photomorphogenesis in Arabidopsis thaliana.

Chloroplast-localized sigma factor (SIG) proteins promote specificity of the plastid-encoded RNA polymerase. SIG2 function appears to be necessary for light-grown Arabidopsis thaliana plants. Specific photoreceptors or light-dependent factors that impact the light-induced accumulation of SIG2 have not been reported. A molecular link between phytochromes and nuclear-encoded SIG2, which impacts photomorphogenesis specifically under red (R) and far-red (FR) light, is described here. Both phyA and phyB promote SIG2 transcript accumulation. Disruption of SIG2 results in R- and FR-specific defects in the inhibition of hypocotyl elongation and cotyledon expansion, although no impairments in these responses are detected for sig2 mutants under blue (B) or white (W) light. SIG2 also impacts root elongation under W and R, and the R-dependent expression of PIF4, encoding a phytochrome-interacting factor, and HY2, which encodes a phytochrome chromophore biosynthetic enzyme. Whereas SIG2 apparently impacts the accumulation of the phytochromobilin (PΦB) phytochrome chromophore, sig2 mutants differ significantly from PΦB mutants, primarily due to wavelength-specific defects in photomorphogenesis and disruption of a distinct subset of phytochrome-dependent responses. The molecular link between phytochromes and SIG2 is likely to be an important part of the co-ordination of gene expression to maintain stoichiometry between the nuclear-encoded phytochrome apoprotein and plastid-derived PΦB, which combine to form photoactive phytochromes, and/or light-dependent SIG2 accumulation is involved in an inductive light signalling pathway co-ordinating components between nucleus and plastids.