The intracellular localization of the florigen FLOWERING LOCUS T (FT) is important for its long-distance transport toward the shoot apical meristem. However, the mechanisms regulating the FT localization remain poorly understood. Here, we discovered that in Arabidopsis thaliana, the chloroplast-localized protein THYLAKOID FORMATION 1 (THF1) physically interacts with FT, sequestering FT in the outer chloroplast envelope. Loss of THF1 function led to temperature-insensitive flowering, resulting in early flowering, especially under low ambient temperatures. THF1 mainly acts in the leaf vasculature and shoot apex to prevent flowering. Mutation of CONSTANS or FT completely suppressed the early flowering of thf1-1 mutants. FT and THF1 interact via their anion binding pocket and coiled-coil domain (CCD), respectively. Deletion of the CCD in THF1 by gene editing caused temperature-insensitive early flowering similar to that observed in the thf1-1 mutant. FT levels in the outer chloroplast envelope decreased in the thf1-1 mutant, suggesting that THF1 is important for sequestering FT. Furthermore, THF1 protein levels decreased in seedlings grown at high ambient temperature, suggesting an explanation for its role in plant responses to ambient temperature. A thf1-1 phosphatidylglycerolphosphate synthase 1 (pgp1) double mutant exhibited additive acceleration of flowering at 23 and 16°C, compared to the single mutants, indicating that THF1 and phosphatidylglycerol (PG) act as independent but synergistic regulators of temperature-responsive flowering. Collectively, our results provide an understanding of the genetic pathway involving THF1 and its role in temperature-responsive flowering and reveal a previously unappreciated additive interplay between THF1 and PG in temperature-responsive flowering.

CONCLUSIONS: Redesigning the N- and C-capping repeats of the native DARPin G3 significantly improved its stability, and may facilitate its purification from the total soluble proteins of high-temperature dried leaf materials of transplastomic plants. Designed ankyrin repeat proteins (DARPins) constitute a promising class of binding molecules that can overcome the limitations of monoclonal antibodies and enable the development of novel therapeutic approaches. Despite their inherent stability, detailed studies have revealed that the original capping repeats derived from natural ankyrin repeat proteins impair the stability of the initial DARPin design. Consequently, the development of thermodynamically stabilized antibody mimetics may facilitate the development of innovative drugs in the future. In this study, we replaced the original N- and C-capping repeats with improved caps to enhance the thermostability of native DARPin G3. Computational analyses suggested that the redesigned thermostable DARPin G3 structure possessed optimal quality and stability. Molecular dynamics simulations verified the stability of the redesigned thermostable DARPin G3 at high temperatures. The redesigned thermostable DARPin G3 was expressed at high levels in tobacco transplastomic plants and subsequently purified from high-temperature dried leaf materials. Thermal denaturation results revealed that the redesigned thermostable DARPin G3 had a higher Tm value than the native DARPin G3, with a Tm of 35.51 °C greater than that of native DARPin G3. The results of the in vitro bioassays confirmed that the purified thermostable DARPin G3 from high-temperature dried leaf materials maintained its binding activity without any loss of affinity and specifically bound to the HER2 receptor on the cell surface. These findings demonstrate the successful improvement in the thermostability of DARPin G3 without compromising its biological activity.

Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region\'s role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.

Chloroplasts play a crucial role in plant defense against pathogens, making them primary targets for pathogen effectors that suppress host immunity. This study characterizes the Plasmopara viticola CRN-like effector, PvCRN20, which interacts with DEG5 in the cytoplasm but not with its interacting protein, DEG8, which is located in the chloroplast. By transiently overexpressing in tobacco leaves, we show that PvCRN20 could inhibit INF1- and Bax-triggered cell death. Constitutive expression of PvCRN20 suppresses the accumulation of reactive oxygen species (ROS) and promotes pathogen colonization. PvCRN20 reduces DEG5 entry into chloroplasts, thereby disrupting DEG5 and DEG8 interactions in chloroplasts. Overexpression of VvDEG5 and VvDEG8 induces ROS accumulation and enhances grapevine resistance to P. viticola, whereas knockout of VvDEG8 represses ROS production and promotes P. viticola colonization. Consistently, ectopic expression of VvDEG5 and VvDEG8 in tobacco promotes chloroplast-derived ROS accumulation, whereas co-expression of PvCRN20 counteracted this promotion by VvDEG5. Therefore, DEG5 is essential for the virulence function of PvCRN20. Although PvCRN20 is located in both the nucleus and cytoplasm, only cytoplasmic PvCRN20 suppresses plant immunity and promotes pathogen infection. Our results reveal that PvCRN20 dampens plant defenses by repressing the chloroplast import of DEG5, thus reducing host ROS accumulation and facilitating pathogen colonization.

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatK\'s preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5\'-splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

Styrax japonicus is a medicinal and ornamental shrub belonging to the Styracaceae family. To explore the diversity and characteristics of the chloroplast genome of S. japonicus, we conducted sequencing and comparison of the chloroplast genomes of four naturally distributed S. japonicus. The results demonstrated that the four chloroplast genomes (157,914-157,962 bp) exhibited a typical quadripartite structure consisting of a large single copy (LSC) region, a small single copy (SSC) region, and a pair of reverse repeats (IRa and IRb), and the structure was highly conserved. DNA polymorphism analysis revealed that three coding genes (infA, psbK, and rpl33) and five intergene regions (petA-psbJ, trnC-petN, trnD-trnY, trnE-trnT, and trnY-trnE) were identified as mutation hotspots. These genetic fragments have the potential to be utilized as DNA barcodes for future identification purposes. When comparing the boundary genes, a small contraction was observed in the IR region of four S. japonicus. Selection pressure analysis indicated positive selection for ycf1 and ndhD. These findings collectively suggest the adaptive evolution of S. japonicus. The phylogenetic structure revealed conflicting relationships among several S. japonicus, indicating divergent evolutionary paths within this species. Our study concludes by uncovering the genetic traits of the chloroplast genome in the differentiation of S. japonicus variety, offering fresh perspectives on the evolutionary lineage of this species.

Cephaleuros species are well-known as plant pathogens that cause red rust or algae spot diseases in many economically cultivated plants that grow in shady and humid environments. Despite their prevalence, the adaptive evolution of these pathogens remains poorly understood. We sequenced and characterized three Cephaleuros (Cephaleuros lagerheimii, Cephaleuros diffusus, and Cephaleuros virescens) chloroplast genomes, and compared them with seven previously reported chloroplast genomes. The chloroplast sequences of C. lagerheimii, C. diffusus, and C. virescens were 480,613 bp, 383,846 bp, and 472,444 bp in length, respectively. These chloroplast genomes encoded 94 genes, including 27 tRNA genes, 3 rRNA genes, and 64 protein-coding genes. Comparative analysis uncovered that the variation in genome size was principally due to the length of intergenic spacer sequences, followed by introns. Furthermore, several highly variable regions (trnY-GTA, trnL-TAG, petA, psbT, trnD-GTC, trnL-TAA, ccsA, petG, psaA, psaB, rps11, rps2, and rps14) were identified. Codon bias analysis revealed that the codon usage pattern of Cephaleuros is predominantly shaped by natural selection. Additionally, six chloroplast protein-coding genes (atpF, chlN, psaA, psaB, psbA, and rbcL) were determined to be under positive selection, suggesting they may play a vital roles in the adaptation of Cephaleuros to low-light intensity habitats.

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5\' and 3\' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5\'UTRs, 10 3\'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5\'atpA, and 3\' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Drought stress is one of the most critical threats to crop productivity and global food security. This review addresses the multiple effects of drought on the process of photosynthesis in major food crops. Affecting both light-dependent and light-independent reactions, drought leads to severe damage to photosystems and blocks the electron transport chain. Plants face a CO2 shortage provoked by stomatal closure, which triggers photorespiration; not only does it reduce carbon fixation efficiency, but it also causes lower overall photosynthetic output. Drought-induced oxidative stress generates reactive oxygen species (ROS) that damage cellular structures, including chloroplasts, further impairing photosynthetic productivity. Plants have evolved a variety of adaptive strategies to alleviate these effects. Non-photochemical quenching (NPQ) mechanisms help dissipate excess light energy as heat, protecting the photosynthetic apparatus under drought conditions. Alternative electron pathways, such as cyclical electron transmission and chloroplast respiration, maintain energy balance and prevent over-reduction of the electron transport chain. Hormones, especially abscisic acid (ABA), ethylene, and cytokinin, modulate stomatal conductance, chlorophyll content, and osmotic adjustment, further increasing the tolerance to drought. Structural adjustments, such as leaf reordering and altered root architecture, also strengthen tolerance. Understanding these complex interactions and adaptive strategies is essential for developing drought-resistant crop varieties and ensuring agricultural sustainability.