Chloroplast Gene Expression Research Articles

The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities.

Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain. Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5' end maturation of mRNAs ascribed to the combined action of 5'→3' exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5'→3' ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-β-lactamase and β-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5'→3' exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5' end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.

Expression of B subunit of E. coli heat-labile enterotoxin in the progenies of transgenic tobacco bred by crossing nuclear- and chloroplast-transgenic lines

The B subunit of Escherichia coli heat-labile toxin (LTB) is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. We previously examined high expression levels of LTB in plants by chloroplast and synthetic LTB gene expression and found substantially higher expression levels of LTB, compared to nuclear LTB expression in wild-type plants. The 2.5% LTB protein of total soluble protein that was observed by chloroplast transformation was approximately 250-fold greater expression than that of LTB via nuclear genome integration. In addition, the amount of LTB protein found in transgenic tobacco leaves using a synthetic LTB gene was 2.2% of the total soluble plant protein, which was approximately 200-fold higher than that in plants with native LTB gene expression. The purpose of our experiment was to increase LTB levels in plants by crossing chloroplast-transformed and synthetic LTB transgenic lines produced previously to express higher LTB levels. LTB protein levels in the F1 transgenic tobacco plants was significantly higher (3.3%), compared to the 2.2% of chloroplast-transformed line or 2.8% of synthetic LTB gene line. Our results suggest that LTB expression was successfully enhanced in the F1 hybrid generation of transgenic tobacco plants.

Zinc Finger Artificial Transcription Factor-Mediated Chloroplast Genome Interrogation in Arabidopsis thaliana.

The large majority of core photosynthesis proteins in plants are encoded by nuclear genes, but a small portion have been retained in the plastid genome. These plastid-encoded chloroplast proteins fulfill essential roles in the process of photochemistry. Here, we report the use of nuclear-encoded, chloroplast-targeted zinc finger artificial transcription factors (ZF-ATFs) with effector domains of prokaryotic origin to modulate the expression of chloroplast genes, and to enhance the photochemical activity and growth characteristics of Arabidopsis thaliana plants. This technique was named chloroplast genome interrogation. Using this novel approach, we obtained evidence that ZF-ATFs can indeed be translocated to chloroplasts of Arabidopsis plants, can modulate their growth and operating light use efficiency of PSII, and finally can induce statistically significant changes in the expression levels of several chloroplast genes. Our data suggest that the distortion of chloroplast gene expression might be a feasible approach to manipulate the efficiency of photosynthesis in plants.

Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.

A role of GUNs-Involved retrograde signaling in regulating Acetyl-CoA carboxylase 2 in Arabidopsis

In Arabidopsis thaliana (Arabidopsis), Acetyl-CoA Carboxylase 2 (ACC2) is a nuclear DNA-encoded and plastid-targeted enzyme that catalyzes the conversion of acetyl-CoA to malonyl-CoA. ACC2 improves plant growth and development when chloroplast translation is impaired. However, little is known about the upstream signals that regulate ACC2. Here, through analyzing the transcriptome changes in brz-insensitive-pale green (bpg) 2-2, a pale-green mutant with impaired chloroplast gene expression resulting from loss of the BPG2 function, we found that the level of ACC2 was significantly up-regulated. Through performing genetic analysis, we further demonstrated that loss of the GENOMES UNCOUPLED 1 (GUN1) or GUN5 function partly perturbed the up-regulation of ACC2 in the bpg2-2 mutant, whereas ABA INSENSITIVE 4 (ABI4)-function-loss had no clear effect on the ACC2 expression. Furthermore, when plants were treated with plastid translation inhibitors, such as lincomycin and spectinomycin, the ACC2 transcriptional level was also markedly increased in a GUN-dependent manner. In conclusion, our results suggested that the GUN-involved plastid-to-nucleus retrograde communication played a role in regulating ACC2 in Arabidopsis.

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.

Effects of cesium accumulation on chlorophyll content and fluorescence of Brassica juncea L.

The aim of our study was to investigate the toxicological mechanism of cesium on Indian mustard (Brassica juncea L.). The impact of cesium toxicity to plants was evaluated using phytophysiology and genetic methods. In this study, Brassica juncea was grown on Cs-contaminated Hoagland's nutrient solution, and chlorophyll content, chlorophyll fluorescence, and Cs bioaccumulation were measured. Transcriptome data was used to perform an in-depth analysis of the molecular mechanisms underlying the effects of Cs accumulation. The results showed that Cs accumulated up to 3586.70 mg kg−1 in B. juncea treated with 100 mg L−1 Cs. The chlorophyll content and several chlorophyll fluorescence parameters (Fv/F0, Fv/Fm, ΦPS II, qP, and NPQ) significantly decreased under Cs exposure. The starting process of PSII was also inhibited under higher Cs conditions. These results indicate that excessive Cs can damage PS II in leaves, decreasing photochemical activity and the energy conversion rate. Further analysis revealed that Cs interfered with the expression of chloroplastic metabolic genes (25 up and 36 down) and inhibited the expression of PsaB, psbC, PetF, LHCA1, and LHCB5. The results indicate that stable Cs leads to abnormal expression of genes related to photosynthesis pathway, blocking the electron transport process from plastoquinone-QA to plastoquinone-QB, resulting in abnormal photosynthesis, which leads to abnormal growth of B. juncea.

Genome uncoupled1 Mutants Are Hypersensitive to Norflurazon and Lincomycin.

Chloroplast development and function relies on the coordinated regulation of chloroplast and nuclear gene expression. Generally, the nucleus is in charge; however, chloroplasts also can signal back to the nucleus via retrograde signaling pathways ([Koussevitzky et al., 2007][1]; [Woodson and Chory,

A nuclear-encoded protein, mTERF6, mediates transcription termination of rpoA polycistron for plastid-encoded RNA polymerase-dependent chloroplast gene expression and chloroplast development

The expression of plastid genes is regulated by two types of DNA-dependent RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). The plastid rpoA polycistron encodes a series of essential chloroplast ribosome subunits and a core subunit of PEP. Despite the functional importance, little is known about the regulation of rpoA polycistron. In this work, we show that mTERF6 directly associates with a 3′-end sequence of rpoA polycistron in vitro and in vivo, and that absence of mTERF6 promotes read-through transcription at this site, indicating that mTERF6 acts as a factor required for termination of plastid genes’ transcription in vivo. In addition, the transcriptions of some essential ribosome subunits encoded by rpoA polycistron and PEP-dependent plastid genes are reduced in the mterf6 knockout mutant. RpoA, a PEP core subunit, accumulates to about 50% that of the wild type in the mutant, where early chloroplast development is impaired. Overall, our functional analyses of mTERF6 provide evidence that it is more likely a factor required for transcription termination of rpoA polycistron, which is essential for chloroplast gene expression and chloroplast development.

Advances in chloroplast expression of recombinant proteins in higher plants

In recent years, gene engineering is developing rapidly and many recombinant proteins have been expressed. The use of plant bioreactor to express specific pharmaceutical proteins provides a new way for the prevention and treatment of some important diseases in human beings. Nowadays, chloroplast genetic transformation and expression system has become a research hotspot in plant bioreactor. Higher plant chloroplasts have unique advantages in the expression of recombinant proteins due to their special structures and inherited characteristics: such as high expression, site-specific integration, and the maternal inheritance characteristics of exogenous genes. The maternal inheritance of chloroplast is helpful for biological safety of transgene escaping by pollens. Many important pharmaceutical proteins have been successfully expressed in plant chloroplasts. As a chloroplast transformation model of higher plants, tobacco has made significant progress in the expression of pharmaceutical proteins, such as vaccine antigens, antibodies, and other important recombinant proteins. Chloroplast genetic transformation in higher plants also provides new techniques and methods for the study of chloroplast gene expression and regulation mechanisms. In order to provide a new idea for the development of chloroplast expression platform and the expression of important pharmaceutical proteins, this review outlined the progress of chloroplast genetic transformation system in higher plants, including the chloroplast transformation principle, vector construction, expression of recombinant proteins and important pharmaceutical proteins, and the effects of recombinant proteins expression on plant metabolism and traits.

Differential impact of heat stress on the expression of chloroplast-encoded genes

Heat shock is one of the major abiotic factors that causes severe retardation in plant growth and development. To dissect the principal effects of hyperthermia on chloroplast gene expression, we studied the temporal dynamics of transcript accumulation for chloroplast-encoded genes in Arabidopsis thaliana and genes for the chloroplast transcription machinery against a background of changes in physiological parameters. A marked reduction in the transcript amounts of the majority of the genes at the early phases of heat shock (HS) was followed by a return to the baseline levels of rbcL and the housekeeping genes clpP, accD, rps14 and rrn16. The decline in the mRNA levels of trnE (for tRNAglu) and the PSI genes psaA and psaB was opposed by the transient increase in the transcript accumulation of ndhF and the PSII genes psbA, psbD, and psbN and their subsequent reduction with the development of stress. However, the up-regulation of PSII genes in response to elevated temperature was absent in the heat stress-sensitive mutants abi1 and abi2 with the impaired degradation of D2 protein. The expression of rpoA and rpoB, which encode subunits of PEP, was strongly down-regulated throughout the duration of the heat treatment. In addition, heat stress-induced PEP deficiency caused the compensatory up-regulation of the genes for the nuclear-encoded RNA polymerases RPOTp and RPOTmp, the PEP-associated proteins PAP6 and PAP8, the Ser/Thr protein kinase cPCK2, and the stress-inducible sigma factor gene SIG5. Thus, heat stress differentially modulates the transcript accumulation of plastid-encoded genes in A. thaliana at least in part via the expression of HS-responsive nuclear genes for the plastid transcription machinery.

Optimising light conditions increases recombinant protein production in Chlamydomonas reinhardtii chloroplasts

The green alga Chlamydomonas reinhardtii provides a platform for cheap, scalable and safe production of complex proteins. Despite the fact that chloroplast gene expression in photosynthetic organisms is tightly regulated by light, most expression studies have analysed chloroplast recombinant protein production under constant light. Here, the influence of light period and intensity on expression of green fluorescent protein (GFP) and a GFP-bacterial-lysin (PlyGBS) fusion protein was analysed. Protein yields were strongly influenced by the light period (6–24 h d−1), the light intensity (0–450 μE m−2 s−1) and trophic condition. Heterotrophic conditions showed low yields of both recombinant proteins due to low growth rates, despite high protein accumulation per cell. Mixotrophic conditions exhibited the highest yields for GFP (4 mg·L−1·d−1) under constant light at 35 μE m−2 s−1 and GFP-PlyGBS (0.4 mg·L−1·d−1) under a light period of 15 h d−1 and 35 μE m−2 s−1. This is due to the high growth rates and cellular protein content. For GFP-PlyGBS the maximum increase in cellular protein accumulation was ~24-fold, and in total protein yield ~10-fold, in comparison to constant light conditions (~200 μE m−2 s−1). The highest yields under photoautrophic conditions were obtained under a 9 h d−1 light period. GFP yielded 1.2 mg·L−1·d−1 and GFP-PlyGBS 0.42 mg·L−1·d−1. This represented a ~5-fold increase in cellular protein accumulation for GFP-PlyGBS in comparison to constant light conditions (~200 μE m−2 s−1). Optimising light conditions to balance growth and protein expression can significantly enhance overall recombinant protein production in C. reinhardtii cultures.

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.

Stabilization and translation of synthetic operon-derived mRNAs in chloroplasts by sequences representing PPR protein-binding sites.

The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5'-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.

SKL1 Is Essential for Chloroplast Development in Arabidopsis.

The Arabidopsis shikimate kinase-like 1 (skl1-8) mutant is characterized by a pigment-defective phenotype. Although the related phenotypical defect mainly has been attributed to the blocking of chloroplast development, the molecular functions of SKL1 remain largely unknown. In this study, we combined multiple approaches to investigate the potential functions of SKL1. Results showed that the skl1-8 mutant exhibited an albino phenotype and had dramatically reduced chlorophyll content as a consequence of a single nuclear recessive gene mutation. Chemical complementation analysis indicated that SKL1 does not function as SK enzyme in the shikimate pathway. In addition, by chlorophyll fluorescence parameters and immunoblot analysis, the levels of photosynthetic proteins are substantially reduced. Moreover, by transcriptome analysis, specific groups of nuclear genes involved in photosynthesis, such as light-harvesting complex, pigment metabolism, carbon metabolism, and chloroplast gene expression, were down-regulated, whereas several defense and oxidative stress responsive genes were up-regulated in the skl1-8 mutant compared with the wide type. Furthermore, we found the expression of genes related to auxin transport and response was repressed in the skl1-8 mutant, probable suggesting that SKL1 is involved in auxin-related pathways during chloroplast development. Together, these results provide a useful reference for characterization of SKL1 function during chloroplast biogenesis and development.

RNA-stabilization factors in chloroplasts of vascular plants.

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.

Green magic: regulation of the chloroplast stress response by (p)ppGpp in plants and algae.

The hyperphosphorylated nucleotides guanosine pentaphosphate and tetraphosphate [together referred to as (p)ppGpp, or 'magic spot'] orchestrate a signalling cascade in bacteria that controls growth under optimal conditions and in response to environmental stress. (p)ppGpp is also found in the chloroplasts of plants and algae where it has also been shown to accumulate in response to abiotic stress. Recent studies suggest that (p)ppGpp is a potent inhibitor of chloroplast gene expression in vivo, and is a significant regulator of chloroplast function that can influence both the growth and the development of plants. However, little is currently known about how (p)ppGpp is wired into eukaryotic signalling pathways, or how it may act to enhance fitness when plants or algae are exposed to environmental stress. This review discusses our current understanding of (p)ppGpp metabolism and its extent in plants and algae, and how (p)ppGpp signalling may be an important factor that is capable of influencing growth and stress acclimation in this major group of organisms.

Differential expression of photosynthesis-related genes in pentaploid interspecific hybrid and its decaploid of Fragaria spp.

Polyploidization always induces a series of changes in genome, transcriptome and epigenetics, of which changes in gene expression are the immediate causes of genotype alterations of polyploid plants. In our previous study on strawberry polyploidization, genes related to photosynthesis were found to undergo changes in gene expression and DNA methylation. Therefore, we chose 11 genes that were closely related to plant photosynthesis and analysed their expression during strawberry hybridization and chromosome doubling. Most genes of pentaploids showed expression levels between parents and were more similar to F. × ananassa. Gene expression levels of decaploids were higher than those of pentaploids and F. × ananassa. Different types of photosynthesis-related genes responded differently to hybridization and chromosome doubling. Chloroplast genes and regulatory genes showed complex responses. Structural genes of the photosynthetic system were expressed at a constant level and displayed a clear dosage effect. The methylation levels of one CG site on SIGE, which regulates expression of chloroplast genes, were negatively correlated with gene expression. In pentaploids and decaploids, more transcripts were from F. × ananassa than from F. viridis. The ratio of transcripts from from F. × ananassa to those from F. viridis was close to the ratio (4:1) of the genome of F. × ananassa to that of F. viridis in pentaploids and decaploids, but there were also some exceptions with obvious deviation.

Bioinformatic Analysis of Chloroplast Gene Expression and RNA Posttranscriptional Maturations Using RNA Sequencing.

Sequencing of total RNA enables the study of the whole plant transcriptome resulting from the simultaneous expression of the three genomes of plant cells (located in the nucleus, mitochondrion and chloroplast). While commonly used for the quantification of the nuclear gene expression, this method remains complex and challenging when applied to organellar genomes and/or when used to quantify posttranscriptional RNA maturations. Here we propose a complete bioinformatical and statistical pipeline to fully characterize the differences in the chloroplast transcriptome between two conditions. Experimental design as well as bioinformatics and statistical analyses are described in order to quantify both gene expression and RNA posttranscriptional maturations, i.e., RNA splicing, editing, and processing, and identify statistically significant differences.

Domain of Unknown Function 143 is required for the functioning of PEP-associated protein DG238 in the chloroplast

Plastid-encoded plastid RNA polymerase (PEP) is essential for chloroplast development and plastid gene expression in Arabidopsis thaliana. However, PEP is a large complex, and many proteins in this complex remain to be identified. We previously reported that Delayed Greening 238 (DG238) interacts with PEP subunit protein FLN1 and may function as a PEP-associated protein and participate in early chloroplast development and PEP-dependent plastid gene expression. DG238 contains Domain of Unknown Function 143 (DUF143), whose function is currently unknown. Here, we found that a deficiency of the DUF143 domain in DG238 affected its localization, which resulted in abnormal interactions with PEP-associated proteins in the chloroplast. Furthermore, DG238 lacking the DUF143 domain or DG238 with only this domain failed to function. Interestingly, the lack of conserved amino acids 193–217 of the DUF143 domain in DG238 also affected its function. In addition to FLN1, DG238 also interacts with other PEP-associated proteins, including FSD2, FSD3, MRL7-L, and MRL7, to regulate plastid gene expression. These results suggest that the DUF143 domain is necessary for the functioning of the PEP-associated protein DG238 in chloroplasts.