Chloroplast Development Research Articles

AcGLK1 promotes chloroplast division through regulating AcFtsZ1 in Actinidia chinensis.

This study unravels a new regulatory member (AcGLK1) that regulates chloroplast division by affecting the expression levels of cytoskeletal filamenting temperature-sensitive Z (FtsZ) in Actinidia chinensis. GOLDEN 2-LIKE (GLK) transcription factor members of GARP subfamily play an irreplaceable role in regulating chloroplast biogenesis and development. Here we report the functional characterization of a novel GLK1 homolog (AcGLK1) isolated from kiwifruit (Actinidia chinensis cultivar 'Hongyang'). Transgenic lines overexpressing AcGLK1 (AcGLK1OE) resulted in an increase of chloroplast number, size and nutrients accumulation in a tomato variety Micro-Tom (Solanum lycopersicum). Transcriptomic data revealed a series of DEGs related to chloroplast division, in which a tomato FtsZ1 homolog (SlFtsZ1) was significantly upregulated in the transgenic lines and could be directly activated by AcGLK1. Furthermore, AcGLK1 was shown to transcriptionally activate expression of kiwifruit FtsZ1 homologous genes (Achv4p23g035689 and Achv4p19g029547) through Y1H and GUS assays. Taken together, we provide evidence showing that AcGLK1 promotes chloroplast division probably through positively regulation of the transcription of FtsZ1 homologs.

SiTCD1, encoding a P-type PPR protein, affects early chloroplast development under low temperature in foxtail millet

SiTCD1, encoding a P-type PPR protein, affects early chloroplast development under low temperature in foxtail millet

Comparative transcriptome and metabolomic analysis reveal key genes and mechanisms responsible for the dark-green leaf color of a strawberry mutant

Photosynthesis is a source of energy for various types of plant life activities and is essential for plant growth and development. Consequently, the study of photosynthetic mechanisms has been a hot spot. Leaf color mutants has always been ideal materials for exploring the mechanisms of chlorophyll metabolism and photosynthesis. In this study, we identified a leaf color mutant of ‘Benihoppe’ strawberry in the field, which exhibited a darker green leaf color compared with the wild type. The content of total chlorophyll and carotenoid in the mutant leaves was elevated by 7.44–20.23% and 8.9–21.92%, respectively, compared with that of the wild type. Additionally, net photosynthetic rate in the mutant increased by 20.13%. Further transcriptome analysis showed that significant upregulation of genes such as GLK1, PPR, and MORF9 in the mutant leaves, which promoted chloroplast development. The expression levels of UROD, PPOC, PORA, CHLG, and CPOX were significantly upregulated during chlorophyll synthesis, while the expression levels of HCAR and CYP89A9 were significantly downregulated during chlorophyll degradation, thus leading to the accumulation of chlorophyll in mutant leaves. The upregulation of gene expression levels such as PetM, AtpD, PGK, and RPI4 during photosynthesis promoted multiple stages of light and dark reaction, thereby enhancing the photosynthetic capacity of the mutant. And the changes in metabolites such as monogalactosyl monoacylglycerol (MGMG), glucuronosyldiacylglycerol (GlcADG), raffinose, etc. also indicate that the mutant has metabolic differences in chloroplast composition and photosynthesis compared to ‘Benihoppe’. The above results not only deepen our understanding of the mechanism behind the dark-green leaf color in strawberry mutants but also provide potential genetic resources for cultivating strawberry varieties with enhanced photosynthetic capacity.

Genome-wide identification and expression analysis of the MORF gene family in celery reveals their potential role in chloroplast development

Chlorophyll is an important nutrient in celery and one of the main indexes of quality evaluation. RNA editing in chloroplasts is an important factor affecting chloroplast development and chlorophyll biosynthesis. Multisite organelle RNA editing factor (MORF) protein is a necessary regulator of chloroplast RNA editing. In this study, a total of 8 MORF genes in celery were identified, which were named AgMORF1a, AgMORF1b, AgMORF2a, AgMORF2b, AgMORF3, AgMORF7, AgMORF8 and AgMORF9 according to their subfamily classification. The physicochemical property, conserved motifs, cis-acting elements and protein interaction were predicted according to the sequences. The phylogenetic relationships and evolutionary selective pressure between MORF genes in celery and other Apiaceae plants were further analyzed. The results showed that AgMORF1b, AgMORF2a, AgMORF2b and AgMORF9 were predicted to be localized in chloroplasts. The evolution of MORF genes in 4 Apiaceae plants including celery, carrot, coriander and water dropwort was influenced by purify selection. Transcriptome data showed that the transcriptional levels of AgMORF2a, AgMORF2b, AgMORF8 and AgMORF9 were relatively higher among all MORF genes in petioles of celery, indicating their major role. RT-qPCR data showed that the expression levels of the above 4 genes were significantly higher in petioles of green celery than those of white celery. This study provided a basis for analyzing the effects of MORF proteins on chloroplast development of celery with different chlorophyll accumulation.

LcASR enhances tolerance to abiotic stress in Leymus chinensis and Arabidopsis thaliana by improving photosynthetic performance.

As a crucial forage grass, Leymus chinensis plays significant roles in soil and water conservation owing to its robust stress resistance. However, the underlying molecular mechanisms of its stress tolerance remain unclear. In this study, a novel gene, designated as LcASR (Abiotic Stress Resistance in Leymus chinensis), imparting resilience to both high light and drought, was identified. Under normal growth conditions, heterologous overexpression of LcASR in Arabidopsis (HO lines) showed no significant difference in appearance compared to wild-type. Nevertheless, HO lines accumulate significantly higher chlorophyll content during the dark-to-light transition compared to the wild-type, indicating that the LcASR protein participates in chlorophyll synthesis during chloroplast development. Meanwhile, transgenic Arabidopsis and L. chinensis plants exhibited resistance to abiotic stresses such as high light and drought. Photosystem complexes analysis revealed that LHCII proteins remained stable within their respective complexes during high light stress. We hypothesize that LcASR may play a role in fine tuning of chlorophyll synthesis to enable plant adaptation to diverse stress conditions. Moreover, overexpression of LcASR in L. chinensis led to agronomically valuable traits such as deeper green color, higher biomass accumulation, prolonged withering period, and extended grazing durations. This study uncovers a novel gene in L. chinensis that enhances forage yield and provides valuable genetic resources for sheepgrass breeding.

Characterization of the complete chloroplast genome and development of molecular markers of Salix

Salix, an economically and ecologically multifunctional tree species widely distributed in China, encompasses five ornamental species sequenced in this study, which are highly beneficial for plant phytoremediation due to their ability to absorb heavy metals. This research utilized high-throughput sequencing to acquire chloroplast genome sequences of Salix, analyzing their gene composition and structural characteristics, identifying potential molecular markers, and laying a foundation for Salix identification and resource classification. Chloroplast DNA was extracted from the leaves of Salix argyracea, Salix dasyclados, Salix eriocephala, Salix integra ‘Hakuro Nishiki’, and Salix suchowensis using an optimized CTAB method. Sequencing was conducted on the Illumina NovaSeq PE150 platform, and bioinformatics tools were employed to compare the structural features and variations within the chloroplast genomes of the Salix. Analysis revealed high similarity among the chloroplast genome sequences of the five Salix species, with a subsequent examination identifying 276, 269, 270, 273, and 273 SSR loci, respectively, along with unique simple repeat sequences in each variety. Comparison of chloroplast genomes across 22 Salix highlighted variations in regions such as matK-trnQ, ndhC-trnV, psbE-petL, rpl36-rps8, and ndhB-rps7, which may serve as valuable molecular markers for willow resource classification studies. In this study, chloroplast genome sequencing and structural analysis of Salix not only enhances the genetic resources of Salix but also forms a critical basis for the development of molecular markers and the exploration of interspecific phylogeny in the genus.

Mutation of rice EARLY LEAF LESION AND SENESCENCE 1(ELS1), which encodes an anthranilate synthase α-subunit, induces ROS accumulation and cell death through activating the tryptophan synthesis pathway in rice.

Lesion-mimic mutants (LMMs) serve as valuable resources for uncovering the molecular mechanisms that govern programmed cell death (PCD) in plants. Despite extensive research, the regulatory mechanisms of PCD and lesion formation in various LMMs remain to be fully elucidated. In this study, we identified a rice LMM named early leaf lesion and senescence 1 (els1), cloned the causal gene through map-based cloning, and confirmed its function through complementation. ELS1 encodes an anthranilate synthase α-subunit involved in anthranilate biosynthesis. It is predominantly localized in chloroplasts and is primarily expressed in light-exposed tissues. Mutation of ELS1 triggers upregulation of its homologous gene, ASA1, via a genetic compensation response, leading to the activation of the tryptophan (Trp) synthesis pathway and amino acid metabolism. The accumulation of abnormal Trp-derived intermediate metabolites results in reactive oxygen species (ROS) production and abnormal PCD in the els1 mutant, ultimately causing the leaf lesion phenotype. The els1 mutant also exhibits reduced chlorophyll content, upregulation of genes related to chloroplast degradation and leaf senescence, and decreased activity of photosynthetic proteins, indicating that ELS1 plays a role in chloroplast development. These factors collectively contribute to the premature leaf senescence observed in the els1 mutant. Our findings shed light on the role of ELS1 in regulating ROS accumulation and PCD in rice, providing further genetic insights into the molecular mechanisms governing leaf lesions and senescence.

Photosynthetic characteristics and genetic mapping of a yellow-green leaf mutant jym165 in soybean

BackgroundLeaves are important sites for photosynthesis and can convert inorganic substances into organic matter. Photosynthetic performance is an important factor affecting crop yield. Leaf colour is closely related to photosynthesis, and leaf colour mutants are considered an ideal material for studying photosynthesis.ResultsWe obtained a yellow-green leaf mutant jym165, using ethyl methane sulfonate (EMS) mutagenesis. Physiological and biochemical analyses indicated that the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the jym165 mutant decreased significantly compared with those in Jiyu47 (JY47). The abnormal chloroplast development of jym165 led to a decrease in net photosynthetic rate and starch content compared with that of JY47. However, quality traits analysis showed that the sum of oil and protein contents in jym165 was higher than that in JY47. In addition, the regional yield (seed spacing: 5 cm) of jym165 increased by 2.42% compared with that of JY47 under high planting density. Comparative transcriptome analysis showed that the yellow-green leaf phenotype was closely related to photosynthesis and starch and sugar metabolism pathways. Genetic analysis suggests that the yellow-green leaf phenotype is controlled by a single recessive nuclear gene. Using Mutmap sequencing, the candidate regions related of leaf colour was narrowed to 3.44 Mb on Chr 10.ConclusionsAbnormal chloroplast development in yellow-green mutants leads to a decrease in the photosynthetic pigment content and net photosynthetic rate, which affects the soybean photosynthesis pathway and starch and sugar metabolism pathways. Moreover, it has the potentiality to increase soybean yield under dense planting conditions. This study provides a useful reference for studying the molecular mechanisms underlying photosynthesis in soybean.

PGL13, a novel allele of OsCRTISO, containing a polyamine oxidase domain, affects chloroplast development and heat stress response in rice

PGL13, a novel allele of OsCRTISO, containing a polyamine oxidase domain, affects chloroplast development and heat stress response in rice

A novel gene silencing strategy based on tobacco rattle virus in Hibiscus mutabilis.

Hibiscus mutabilis L. is a popular regional characteristic plant in China, cultivated for its attractive flower colors, extended bloom time, and medicinal properties. To enhance molecular breeding and gene function studies, we conducted transcriptome analysis and identified valuable genes in previous research. Nonetheless, the current inefficient and labor-intensive transformation techniques have hindered their applications. Virus-induced gene silencing (VIGS) provides a precise and effective strategy for post-transcriptional down-regulation of endogenous gene expression. We investigated the performance of tobacco rattle virus (TRV) as a tool for targeting and silencing the gene encoding the protein involved in chloroplast development, cloroplastos alterados 1 (altered chloroplast; CLA1), of H. mutabilis through Agrobacterium tumefaciens-mediated infiltration. By effectively suppressing the CLA1 gene associated with chloroplast development in H. mutabilis via the TRV-VIGS system, we have illustrated the inaugural implementation of VIGS in this species. Quantitative RT-PCR proved that HmCLA1 expression in agro-infiltrated plants was lower than in the mock-infiltrated (mock) and the control (CK) plants. Phenotypic observations corroborated the albino phenotype in leaves following successful HmCLA1 silencing. Our study showcases TRV-VIGS as a potential gene silencing tool for H. mutabilis, facilitating functional genomics studies and molecular breeding efforts in this species.

The role of SlCHRC in carotenoid biosynthesis and plastid development in tomato fruit

Chromoplasts are specialized plastids in plants involved in carotenoid synthesis, accumulation, and stress resistance. In tomatoes (Solanum lycopersicum), the Chromoplast-associated carotenoid binding protein (CHRC) regulates chromoplast development and carotenoid accumulation, although its precise mechanisms are not yet fully understood. To investigate the role of SlCHRC in carotenoid biosynthesis, we generated transgenic tomatoes using overexpression (oe-SlCHRC) and CRISPR/Cas9-mediated gene editing (cr-SlCHRC) techniques. The results demonstrated inhibited fruit ripening and delayed onset of color break in both transgenic lines. The oe-SlCHRC lines exhibited increased carotenoid accumulation, particularly (E/Z)-phytoene, lycopene, and γ-carotene, with abundant plastoglobules and carotenoid crystals observed via TEM. In contrast, cr-SlCHRC mutants showed a greener phenotype, reduced carotenoid content, and fewer plastoglobules at the BK + 10 stage. Transcriptome analysis indicated that SlCHRC influences key genes in carotenoid biosynthesis, such as SlNCED2, as well as genes related to chloroplast development, photosynthesis, and plastoglobule formation. Additionally, SlCHRC enhances heat stress tolerance in tomato fruits by upregulating heat shock proteins (HSPs), antioxidants, and proline accumulation. These findings indicate that SlCHRC plays a crucial role in improving tomato fruit quality under heat stress conditions.

Transcriptional and genetic characteristic of chimera pea generation via double ethyl methanesulfonate-induced mutation revealed by transcription analysis.

Ethyl methanesulfonate (EMS)-induced mutagenesis is a prominent method for generating plant mutants, often resulting in chimera plants; however, their transcriptional and genetic characteristic remain elusive. In this investigation, chimera pea (Pisum sativum L.) specimens, labeled GY1 and GY2, exhibiting a distinctive phenotype with yellow and green leaves were meticulously cultivated via sequential double EMS mutagenesis. The observed color disparity between the yellow and green leaves was attributed to a significant reduction in chlorophyll content coupled with heightened lutein levels in both chimeric variants. Transcriptome profiling revealed the enrichment of differentially expressed genes in both GY1 and GY2, specifically implicating Kyoto Encyclopedia of Genes and Genomes pathways linked to amino acid biosynthesis and ribosome development, alongside Gene Ontology (GO) biological processes linked with stress response mechanisms. Few structural genes associated with chlorophyll and lutein biosynthesis exhibited discernible differential expression. Despite these functional similarities, distinctive nuances were evident between specimens, with GY1 exhibiting enrichment in GO pathways related to chloroplast development and GY2 showing enrichment for ribosome development pathways. Single-nucleotide polymorphism (SNP) analysis uncovered a shared pool of 599 and 598 polymorphisms in the yellow and green leaves of GY1 and GY2, respectively, likely stemming from the initial EMS mutagenesis step. Further investigation revealed an increased number of unique SNPs in the yellow leaves following the second EMS application, whereas the green leaves exhibited sparse and unique SNP occurrences, suggestive of potential evasion from secondary mutagenesis. This inherent genetic variability underpins the mechanism underlying the formation of chimera plants. Predominant base mutations induced by EMS were characterized by G/A and C/T transitions, constituting 74.1% of the total mutations, aligning with established EMS mutation induction paradigms. Notably, genes encoding the eukaryotic translation initiation factor eIIso4G and the ubiquitin ligase RKP, known to modulate leaf color in model plants, harbored two SNPs in the yellow leaves of both GY1 and GY2, implicating their putative role in the yellow leaf phenotype. Collectively, this study provides novel insights into the transcriptional and genetic characteristics of chimera plants via EMS-induced mutagenesis.

Conjunctive BSA-Seq and BSR-Seq to Map the Genes of Yellow Leaf Mutations in Hot Peppers (Capsicum annuum L.).

Yellow leaf mutations have been widely used to study the chloroplast structures, the pigment synthesis, the photosynthesis mechanisms and the chlorophyll biosynthesis pathways across various species. For this study, a spontaneous mutant with the yellow leaf color named 96-140YBM was employed to explore the primary genetic elements that lead to the variations in the leaf color of hot peppers. To identify the pathways and genes associated with yellow leaf phenotypes, we applied sequencing-based Bulked Segregant Analysis (BSA-Seq) combined with BSR-Seq. We identified 4167 differentially expressed genes (DEGs) in the mutant pool compared with the wild-type pool. The results indicated that DEGs were involved in zeatin biosynthesis, plant hormone signal transduction, signal transduction mechanisms, post-translational modification and protein turnover. A total of 437 candidates were identified by the BSA-Seq, while the BSR-Seq pinpointed four candidate regions in chromosomes 8 and 9, containing 222 candidate genes. Additionally, the combination of BSA-Seq and BSR-Seq showed that there were 113 overlapping candidate genes between the two methods, among which 8 common candidates have been previously reported to be related to the development of chloroplasts, the photomorphogenesis and chlorophyll formation of plant chloroplasts and chlorophyll biogenesis. qRT-PCR analysis of the 8 common candidates showed higher expression levels in the mutant pool compared with the wild-type pool. Among the overlapping candidates, the DEG analysis showed that the CaKAS2 and CaMPH2 genes were down-regulated in the mutant pool compared to the wild type, suggesting that these genes may be key contributors to the yellow leaf phenotype of 96-140YBM. This research will deepen our understanding of the genetic basis of leaf color formation and provide valuable information for the breeding of hot peppers with diverse leaf colors.

SPL50 Regulates Cell Death and Resistance to Magnaporthe Oryzae in Rice

BackgroundThe identification of spotted leaf 50 (spl50), a novel lesion mimic mutant (LMM) in rice, provides critical insights into the mechanisms underlying programmed cell death (PCD) and innate immunity in plants.ResultsBased on ethyl methane sulfonate (EMS)-induced mutagenesis, the spl50 mutant mimics hypersensitive responses in the absence of pathogen by displaying spontaneous necrotic lesions after the tillering phase. SPL50, an ARM repeat protein essential for controlling reactive oxygen species (ROS) metabolism and boosting resistance to blast disease, was identified by map-based cloning techniques. This work also demonstrates the detrimental effects of spl50 on photosynthetic efficiency and chloroplast development. The crucial significance of SPL50 in cellular signaling and stress response is shown by its localization to the cytoplasm and constitutive expression in various plant tissues. In light of growing concerns regarding global food security, this study highlights the pivotal role of SPL50 in regulating programmed cell death (PCD) and enhancing the immune response in plants, contributing to strategies for improving crop disease resistance.ConclusionsThe novel identification of the SPL50 gene in rice, encoding an ARM repeat protein, reveals its pivotal role in regulating PCD and innate immune responses independently of pathogen attack.

The pseudo-type response regulator gene Clsc regulates rind stripe coloration in watermelon1

The color and pattern of watermelon's rind are crucial external traits that directly affect consumer preferences. Watermelons with stripes having a stronger color than the background rind are ideal for studying plant stripes, while there is still limited knowledge about the genetic mechanisms underlying stripe coloration due to the lack of germplasm resources. In this study, we focused on a watermelon germplasm with colorless stripes, and genetic analysis revealed that the trait is controlled by a single recessive gene. The gene Clsc (Citrullus lanatus stripe coloration), which is responsible for the colorless stripe, was confined into a 147.6 kb region on chromosome 9 by linkage analysis in a large F2 mapping population. Further analysis revealed that the Cla97C09G175170 gene, encoding the APRR2 transcription factor, plays a crucial role in determining watermelon's colorless stripe phenotype and was deduced to be related to chlorophyll and chloroplast development. Based on the physiology experiment results, it can be inferred that Cla97C09G175170 may significantly influence chloroplast development and chlorophyll synthesis in watermelon. The results of this study will be helpful for better understanding the molecular mechanism of stripe coloration in watermelon and for the development of marker-assisted selection (MAS) for new watermelon cultivars.

The yellow-cotyledon gene (ATYCO) is a crucial factor for thylakoid formation and photosynthesis regulation in Arabidopsis

Chloroplast development underpins plant growth, by facilitating not only photosynthesis but also other essential biochemical processes. Nonetheless, the regulatory mechanisms and functional components of chloroplast development remain largely uncharacterized due to their complexity. In our study, we identified a plastid-targeted gene, ATYCO/RP8/CDB1, as a critical factor in early chloroplast development in Arabidopsis thaliana. YCO knock-out mutant (yco) exhibited a seedling-lethal, albino phenotype, resulting from dysfunctional chloroplasts lacking thylakoid membranes. Conversely, YCO knock-down mutants produced a chlorophyll-deficient cotyledon and normal leaves when supplemented with sucrose. Transcription analysis also revealed that YCO deficiency could be partially compensated by sucrose supplementation, and that YCO played different roles in the cotyledons and the true leaves. In YCO knock-down mutants, the transcript levels of plastid-encoded RNA polymerase (PEP)-dependent genes and nuclear-encoded photosynthetic genes, as well as the accumulation of photosynthetic proteins, were significantly reduced in the cotyledons. Moreover, the chlorophyll-deficient phenotype in YCO knock-down line can be effectively suppressed by inhibition of PSI cyclic electron transport activity, implying an interaction between YCO and PSI cyclic electron transport. Taken together, our findings de underscore the vital role of YCO in early chloroplast development and photosynthesis.

The Rice YL4 Gene Encoding a Ribosome Maturation Domain Protein Is Essential for Chloroplast Development.

Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits.

MYB-related transcription factors control chloroplast biogenesis

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors. However, glk mutants contain residual chlorophyll, indicating that other proteins must be involved. Here, we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana. In both species, double-mutant alleles in MYB-related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in GLK mutants. Genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins, whereas those allowing CO2 fixation, photorespiration, and photosystem assembly and repair require MYB-related proteins. Regulation between the MYB-related and GLK transcription factors appears more extensive in A.thaliana than in M.polymorpha. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

A chromosome-level genome assembly for the paramylon-producing microalga Euglena gracilis

Euglena gracilis (E. gracilis), pivotal in the study of photosynthesis, endosymbiosis, and chloroplast development, is also an industrial microalga for paramylon production. Despite its importance, E. gracilis genome exploration faces challenges due to its intricate nature. In this study, we achieved a chromosome-level de novo assembly (2.37 Gb) using Illumina, PacBio, Bionano, and Hi-C data. The assembly exhibited a contig N50 of 619 Kb and scaffold N50 of 1.12 Mb, indicating superior continuity. Approximately 99.83% of the genome was anchored to 46 chromosomes, revealing structural insights. Repetitive elements constituted 58.84% of the sequences. Functional annotations were assigned to 39,362 proteins, enhancing interpretative power. BUSCO analysis confirmed assembly completeness at 80.39%. This first high-quality E. gracilis genome offers insights for genetics and genomics studies, overcoming previous limitations. The impact extends to academic and industrial research, providing a foundational resource.

Membrane association of active genes organizes the chloroplast nucleoid structure

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.