Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.

Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.

CRUMPLED LEAF supports plastid OUTER ENVELOPE PROTEIN OF 80 KDA complex formation in Arabidopsis.

Embedded ß-barrel proteins in the outer envelope membrane mediate most cellular trafficking between the cytoplasm and plastids. Although the TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 75-V (TOC75-V)/OUTER ENVELOPE PROTEIN OF 80 KDA (OEP80) complex has been implicated in the insertion and assembly of ß-barrel proteins in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts, relatively little is known about this process. CRUMPLED LEAF (CRL) encodes a chloroplast outer envelope membrane-localized protein, and its loss-of-function mutation results in pleiotropic defects, including altered plant morphogenesis, growth retardation, suppression of plastid division, and spontaneous light intensity-dependent localized cell death. A suppressor screen conducted on mutagenized crl mutants revealed that a missense mutation in OEP80 suppresses the pleiotropic defects of crl. Furthermore, we found that OEP80 complex formation is compromised in crl. Additionally, we demonstrated that CRL interacts with OEP80 in vivo and that a portion of CRL is present at the same molecular weight as the OEP80 complex. Our results suggest that CRL interacts with OEP80 to facilitate its complex formation. CRL is involved in plastid protein import; therefore, the pleiotropic defects in crl are likely due to the combined effects of decreased plastid protein import and altered membrane integration of ß-barrel proteins in the outer envelope membrane. This study sheds light on the mechanisms that allow ß-barrel protein integration into the plastid outer envelope membrane and the importance of this finding for plant cellular processes.

Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aß) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aß aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aß aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aß aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aß aggregation and determined that the chaperone prevents Aß aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aß in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aß aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aß aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aß peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Novel Plastid Genome Characteristics in Fugacium kawagutii and the Trend of Accelerated Evolution of Plastid Proteins in Dinoflagellates.

Typical (peridinin-containing) dinoflagellates possess plastid genomes composed of small plasmids named "minicircles". Despite the ecological importance of dinoflagellate photosynthesis in corals and marine ecosystems, the structural characteristics, replication dynamics, and evolutionary forcing of dinoflagellate plastid genomes remain poorly understood. Here, we sequenced the plastid genome of the symbiodiniacean species Fugacium kawagutii and conducted comparative analyses. We identified psbT-coding minicircles, features previously not found in Symbiodiniaceae. The copy number of F. kawagutii minicircles showed a strong diel dynamics, changing between 3.89 and 34.3 copies/cell and peaking in mid-light period. We found that F. kawagutii minicircles are the shortest among all dinoflagellates examined to date. Besides, the core regions of the minicircles are highly conserved within genus in Symbiodiniaceae. Furthermore, the codon usage bias of the plastid genomes in Heterocapsaceae, Amphidiniaceae, and Prorocentraceae species are greatly influenced by selection pressure, and in Pyrocystaceae, Symbiodiniaceae, Peridiniaceae, and Ceratiaceae species are influenced by both natural selection pressure and mutation pressure, indicating a family-level distinction in codon usage evolution in dinoflagellates. Phylogenetic analysis using 12 plastid-encoded proteins and five nucleus-encoded plastid proteins revealed accelerated evolution trend of both plastid- and nucleus-encoded plastid proteins in peridinin- and fucoxanthin-dinoflagellate plastids compared to plastid proteins of nondinoflagellate algae. These findings shed new light on the structure and evolution of plastid genomes in dinoflagellates, which will facilitate further studies on the evolutionary forcing and function of the diverse dinoflagellate plastids. The accelerated evolution documented here suggests plastid-encoded sequences are potentially useful for resolving closely related dinoflagellates.

Light-induced LLPS of the CRY2/SPA1/FIO1 complex regulating mRNA methylation and chlorophyll homeostasis in Arabidopsis.

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m6A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid-liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis.

Interaction of PALE CRESS with PAP2/pTAC2 and PAP3/pTAC10 affects the accumulation of plastid-encoded RNA polymerase complexes in Arabidopsis.

The transcription of photosynthesis genes in chloroplasts is largely mediated by the plastid-encoded RNA polymerase (PEP), which resembles prokaryotic-type RNA polymerases, but with plant-specific accessory subunits known as plastid transcriptionally active chromosome proteins (pTACs) or PEP-associated proteins (PAPs). However, whether additional factors are involved in the biogenesis of PEP complexes remains unknown. Here, we investigated the function of an essential gene, PALE CRESS (PAC), in the accumulation of PEP complexes in chloroplasts. We established that an Arabidopsis leaf variegation mutant, variegated 6-1 (var6-1), is a hypomorphic allele of PAC. Unexpectedly, we revealed that a fraction of VAR6/PAC is associated with thylakoid membranes, where it interacts with PEP complexes. The accumulation of PEP complexes is defective in both var6-1 and the null allele var6-2. Further protein interaction assays confirmed that VAR6/PAC interacts directly with the PAP2/pTAC2 and PAP3/pTAC10 subunits of PEP complexes. Moreover, we generated viable hypomorphic alleles of the essential gene PAP2/pTAC2, and revealed a genetic interaction between PAC and PAP2/pTAC2 in photosynthesis gene expression and PEP complex accumulation. Our findings establish that VAR6/PAC affects PEP complex accumulation through interactions with PAP2/pTAC2 and PAP3/pTAC10, and provide new insights into the accumulation of PEP and chloroplast development.

Relocation of chloroplast proteins from cytosols into chloroplasts.

The chloroplasts in terrestrial plants play a functional role as a major sensor for perceiving physiological changes under normal and stressful conditions. Despite the fact that the plant chloroplast genome encodes around 120 genes, which are mainly essential for photosynthesis and chloroplast biogenesis, the functional roles of the genes remain to be determined in plant's response to environmental stresses. Photosynthetic electron transfer D (PETD) is a key component of the chloroplast cytochrome b6f complex. Chloroplast ndhA (NADH dehydrogenase A) and ndhB (NADH dehydrogenase B) interact with photosystem I (PSI), forming NDH-PSI supercomplex. Notably, artificial targeting of chloroplasts-encoded proteins, PETD, NDHA, or NDHB, was successfully relocated from cytosols into chloroplasts. The result suggests that artificial targeting of proteins to chloroplasts is potentially open to the possibility of chloroplast biotechnology in engineering of plant tolerance against biotic and abiotic stresses.

Chloroplast protein translocation pathways and ubiquitin-dependent regulation at a glance.

Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.

The chloroplast protein HCF164 is predicted to be associated with Coffea SH9 resistance factor against Hemileia vastatrix.

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid "Híbrido de Timor". Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Competition co-immunoprecipitation reveals the interactors of the chloroplast CPN60 chaperonin machinery.

The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

Chloroplast-related host proteins interact with NIb and NIa-Pro of soybeans mosaic virus and induce resistance in the susceptible cultivar.

To gain a deeper understanding of the molecular mechanisms involved in viral infection and the corresponding plant resistance responses, it is essential to investigate the interactions between viral and host proteins. In the case of viral infections in plants, a significant portion of the affected gene products are closely associated with chloroplasts and photosynthesis. However, the molecular mechanisms underlying the interplay between the virus and host chloroplast proteins during replication remain poorly understood. In our previous study, we made an interesting discovery regarding soybean mosaic virus (SMV) infection in resistant and susceptible soybean cultivars. We found that the photosystem I (PSI) subunit (PSaC) and ATP synthase subunit α (ATPsyn-α) genes were up-regulated in the resistant cultivar following SMV-G7H and SMV-G5H infections compared to the susceptible cultivar. Overexpression of these two genes within the SMV-G7H genome in the susceptible cultivar Lee74 (rsv3-null) reduced SMV accumulation, whereas silencing of the PSaC and ATPsyn-α genes promoted SMV accumulation. We have also found that the PSaC and ATPsyn-α proteins are present in the chloroplast envelope, nucleus, and cytoplasm. Building on these findings, we now characterized protein-protein interactions between PSaC and ATPsyn-α with two viral proteins, NIb and NIa-Pro, respectively, of SMV. Through co-immunoprecipitation (Co-IP) experiments, we confirmed the interactions between these proteins. Moreover, when the C-terminal region of either PSaC or ATPsyn-α was overexpressed in the SMV-G7H genome, we observed a reduction in viral accumulation and systemic infection in the susceptible cultivar. Based on these results, we propose that the PSaC and ATPsyn-α genes play a modulatory role in conferring resistance to SMV infection by influencing the function of NIb and NIa-Pro-in SMV replication and movement. The identification of these photosynthesis-related genes as key players in the interplay between the virus and the host provides valuable insights for developing more targeted control strategies against SMV. Additionally, by utilizing these genes, it may be possible to genetically engineer plants with improved photosynthetic efficiency and enhanced resistance to SMV infection.

A Machine Learning Framework Identifies Plastid-Encoded Proteins Harboring C3 and C4 Distinguishing Sequence Information.

C4 photosynthesis is known to have at least 61 independent origins across plant lineages making it one of the most notable examples of convergent evolution. Of the >60 independent origins, a predicted 22-24 origins, encompassing greater than 50% of all known C4 species, exist within the Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae (PACMAD) clade of the Poaceae family. This clade is therefore primed with species ideal for the study of genomic changes associated with the acquisition of the C4 photosynthetic trait. In this study, we take advantage of the growing availability of sequenced plastid genomes and employ a machine learning (ML) approach to screen for plastid genes harboring C3 and C4 distinguishing information in PACMAD species. We demonstrate that certain plastid-encoded protein sequences possess distinguishing and informative sequence information that allows them to train accurate ML C3/C4 classification models. Our RbcL-trained model, for example, informs a C3/C4 classifier with greater than 99% accuracy. Accurate prediction of photosynthetic type from individual sequences suggests biologically relevant, and potentially differing roles of these sequence products in C3 versus C4 metabolism. With this ML framework, we have identified several key sequences and sites that are most predictive of C3/C4 status, including RbcL, subunits of the NAD(P)H dehydrogenase complex, and specific residues within, further highlighting their potential significance in the evolution and/or maintenance of C4 photosynthetic machinery. This general approach can be applied to uncover intricate associations between other similar genotype-phenotype relationships.

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling.

The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis.

cpSRP43 Is Both Highly Flexible and Stable: Structural Insights Using a Combined Experimental and Computational Approach.

The novel multidomain protein, cpSRP43, is a unique subunit of the post-translational chloroplast signal recognition particle (cpSRP) targeting pathway in higher plants. The cpSRP pathway is responsible for targeting and insertion of light-harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. Upon emergence into the stroma, LHCPs form a soluble transit complex with the cpSRP heterodimer, which is composed of cpSRP43 and cpSRP54. cpSRP43 is irreplaceable as a chaperone to LHCPs in their translocation to the thylakoid membrane and remarkable in its ability to dissolve aggregates of LHCPs without the need for external energy input. In previous studies, cpSRP43 has demonstrated significant flexibility and interdomain dynamics. In this study, we explore the structural stability and flexibility of cpSRP43 using a combination of computational and experimental techniques and find that this protein is concurrently highly stable and flexible. In addition to microsecond-level unbiased molecular dynamics (MD), biased MD simulations based on system-specific collective variables are used along with biophysical experimentation to explain the basis of the flexibility and stability of cpSRP43, showing that the free and cpSRP54-bound cpSRP43 has substantially different conformations and conformational dynamics.

Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.

Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.

STAY-GREEN Accelerates Chlorophyll Degradation in Magnolia sinostellata under the Condition of Light Deficiency.

Species of the Magnoliaceae family are valued for their ornamental qualities and are widely used in landscaping worldwide. However, many of these species are endangered in their natural environments, often due to being overshadowed by overstory canopies. The molecular mechanisms of Magnolia's sensitivity to shade have remained hitherto obscure. Our study sheds light on this conundrum by identifying critical genes involved in governing the plant's response to a light deficiency (LD) environment. In response to LD stress, Magnolia sinostellata leaves were endowed with a drastic dwindling in chlorophyll content, which was concomitant to the downregulation of the chlorophyll biosynthesis pathway and upregulation in the chlorophyll degradation pathway. The STAY-GREEN (MsSGR) gene was one of the most up-regulated genes, which was specifically localized in chloroplasts, and its overexpression in Arabidopsis and tobacco accelerated chlorophyll degradation. Sequence analysis of the MsSGR promoter revealed that it contains multiple phytohormone-responsive and light-responsive cis-acting elements and was activated by LD stress. A yeast two-hybrid analysis resulted in the identification of 24 proteins that putatively interact with MsSGR, among which eight were chloroplast-localized proteins that were significantly responsive to LD. Our findings demonstrate that light deficiency increases the expression of MsSGR, which in turn regulates chlorophyll degradation and interacts with multiple proteins to form a molecular cascade. Overall, our work has uncovered the mechanism by which MsSGR mediates chlorophyll degradation under LD stress conditions, providing insight into the molecular interactions network of MsSGR and contributing to a theoretical framework for understanding the endangerment of wild Magnoliaceae species.

Prediction of protein subplastid localization and origin with PlastoGram.

Due to their complex history, plastids possess proteins encoded in the nuclear and plastid genome. Moreover, these proteins localize to various subplastid compartments. Since protein localization is associated with its function, prediction of subplastid localization is one of the most important steps in plastid protein annotation, providing insight into their potential function. Therefore, we create a novel manually curated data set of plastid proteins and build an ensemble model for prediction of protein subplastid localization. Moreover, we discuss problems associated with the task, e.g. data set sizes and homology reduction. PlastoGram classifies proteins as nuclear- or plastid-encoded and predicts their localization considering: envelope, stroma, thylakoid membrane or thylakoid lumen; for the latter, the import pathway is also predicted. We also provide an additional function to differentiate nuclear-encoded inner and outer membrane proteins. PlastoGram is available as a web server at https://biogenies.info/PlastoGram and as an R package at https://github.com/BioGenies/PlastoGram . The code used for described analyses is available at https://github.com/BioGenies/PlastoGram-analysis .

The novel chloroplast glucose transporter pGlcT2 affects adaptation to extended light periods.

Intracellular sugar compartmentation is critical in plant development and acclimation to challenging environmental conditions. Sugar transport proteins are present in plasma membranes and in membranes of organelles such as vacuoles, the Golgi apparatus, and plastids. However, there may exist other transport proteins with uncharacterized roles in sugar compartmentation. Here we report one such novel transporter of the Monosaccharide Transporter Family, the closest phylogenetic homolog of which is the chloroplast-localized glucose transporter pGlcT and that we therefore term plastidic glucose transporter 2 (pGlcT2). We show, using gene-complemented glucose uptake deficiency of an Escherichia coli ptsG/manXYZ mutant strain and biochemical characterization, that this protein specifically facilitates glucose transport, whereas other sugars do not serve as substrates. In addition, we demonstrate pGlcT2-GFP localized to the chloroplast envelope and that pGlcT2 is mainly produced in seedlings and in the rosette center of mature Arabidopsis plants. Therefore, in conjunction with molecular and metabolic data, we propose pGlcT2 acts as a glucose importer that can limit cytosolic glucose availability in developing pGlcT2-overexpressing seedlings. Finally, we show both overexpression and deletion of pGlcT2 resulted in impaired growth efficiency under long day and continuous light conditions, suggesting pGlcT2 contributes to a release of glucose derived from starch mobilization late in the light phase. Together, these data indicate the facilitator pGlcT2 changes the direction in which it transports glucose during plant development and suggest the activity of pGlcT2 must be controlled spatially and temporarily in order to prevent developmental defects during adaptation to periods of extended light.