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The expression profiles of the PAP genes, encoding proteins associated with plastid multisubunit RNA polymerase, were studied in dry seeds, during germination, and at the early stages of Arabidopsis thaliana seedling formation. A detailed analysis of the PAP transcript levels by RT-PCR showed that the transition of seeds from dormancy to active growth is accompanied by a drastic increase in the transcript accumulation of all studied genes on the first day of germination, both in the light and in the dark. Further changes in transcript levels differed among PAP genes and were apparently determined by their functional specificity. It was established for the first time that the expression of individual PAP genes is regulated by circadian rhythms, in addition to factors of ontogenetic and light nature.

The high-value carotenoid astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type–like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid., Inducible expression of a synthetic astaxanthin operon in plastids alleviates the growth phenotype of constitutive pathway expression and provides insights into carotenoid biosynthesis bottlenecks.

Key message Phosphoglycerate Dehydrogenase 1 of the phosphorylated pathway of serine biosynthesis, active in heterotrophic plastids, is required for the synthesis of serine to enable plant growth at high rates of indolic glucosinolate biosynthesis. Abstract Plants have evolved effective strategies to defend against various types of pathogens. The synthesis of a multitude of specialized metabolites represents one effective approach to keep plant attackers in check. The synthesis of those defense compounds is cost intensive and requires extensive interaction with primary metabolism. However, how primary metabolism is adjusted to fulfill the requirements of specialized metabolism is still not completely resolved. Here, we studied the role of the phosphorylated pathway of serine biosynthesis (PPSB) for the synthesis of glucosinolates, the main class of defensive compounds in the model plant Arabidopsis thaliana. We show that major genes of the PPSB are co-expressed with genes required for the synthesis of tryptophan, the unique precursor for the formation of indolic glucosinolates (IG). Transcriptional and metabolic characterization of loss-of-function and dominant mutants of ALTERED TRYPTOPHAN1-like transcription factors revealed demand driven activation of PPSB genes by major regulators of IG biosynthesis. Trans-activation of PPSB promoters by ATR1/MYB34 transcription factor in cultured root cells confirmed this finding. The content of IGs were significantly reduced in plants compromised in the PPSB and these plants showed higher sensitivity against treatment with 5-methyl-tryptophan, a characteristic behavior of mutants impaired in IG biosynthesis. We further found that serine produced by the PPSB is required to enable plant growth under conditions of high demand for IG. In addition, PPSB-deficient plants lack the growth promoting effect resulting from interaction with the beneficial root-colonizing fungus Colletotrichum tofieldiae.

Pentatricopeptide repeat (PPR) proteins play important roles in the post-transcriptional modification of organellar RNAs in plants. However, the function of most PPR proteins remains unknown. Here, we characterized the rice (Oryza sativa L.) chlorophyll deficient 4 (cde4) mutant which exhibits an albino phenotype during early leaf development, with decreased chlorophyll contents and abnormal chloroplasts at low-temperature (20°C). Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts. In the cde4 mutant, plastid-encoded polymerase (PEP)-dependent transcript levels were significantly reduced, but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20°C. CDE4 directly binds to the transcripts of the chloroplast genes rpl2, ndhA, and ndhB. Intron splicing of these transcripts was defective in the cde4 mutant at 20°C, but was normal at 32°C. Moreover, CDE4 interacts with the guanylate kinase VIRESCENT 2 (V2); overexpression of V2 enhanced CDE4 protein stability, thereby rescuing the cde4 phenotype at 20°C. Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under low-temperature conditions.

Photosystem II (PS II) of oxygenic photosynthesis is found in the thylakoid membranes of plastids and cyanobacteria. The mature PS II complex comprises a central core of four membrane proteins that bind the majority of the redox-active cofactors. In cyanobacteria the central core is surrounded by 13 low-molecular-weight (LMW) subunits which each consist of one or two transmembrane helices. Three additional hydrophilic subunits known as PsbO, PsbU and PsbV are found associated with hydrophilic loops belonging to the core proteins protruding into the thylakoid lumen. During biogenesis the majority of the LMW subunits are known to initially associate with individual pre-assembly complexes consisting of one or more of the core proteins; however, the point at which the PsbJ LMW subunit binds to PS II is not known. The majority of models for PS II biogenesis propose that the three extrinsic proteins and PsbJ bind in the final stages of PS II assembly. We have investigated the impact of creating the double mutants ∆PsbJ:∆PsbO, ∆PsbJ:∆PsbU and ∆PsbJ:∆PsbV to investigate potential cooperation between these subunits in the final stages of biogenesis. Our results indicate that PsbJ can bind to PS II in the absence of any one of the extrinsic proteins. However, unlike their respective single mutants, the ∆PsbJ:∆PsbO and ∆PsbJ:∆PsbV strains were not photoautotrophic and were unable to support oxygen evolution suggesting a functional oxygen-evolving complex could not assemble in these strains. In contrast, the PS II centers formed in the ∆PsbJ:∆PsbU strain were capable of photoautotrophic growth and could support oxygen evolution when whole-chain electron transport was supported by the addition of bicarbonate.

The common carbon and nitrogen currency, 2-oxoglutarate, could become a valuable resource for nitrogen assimilation and carbon centered biochemical fates. Here in this in silico study, a myriad of factors was used, namely phylogeny, sequence comparisons, and presence and location of clustered cysteines in specific plastid transporters of 2-oxoglutarate, to examine their evolution away from more generalized transporters. This transition would be to adopt the capability of internalizing 2-oxoglutarate alone or with superior specificities at the expense of malate. In phylogeny, the specific 2-oxoglutarate transporters (Cluster 1) are clustered in a separate clade away from 2 clades of general transporters (Cluster 2 and 3). The exclusivity (Cluster 1) and promiscuity of transporters (Cluster 2 and 3) compared to Arabidopsis counterparts characterized prior to this study, were used as a benchmark for my study. Within this mother clade of exclusive transporters, C4 and C3 2-oxoglutarate transporters once again form separate clusters of monophyly. Furthermore, a pattern of Cys –X-X-Cys-X(19)-Cys is conserved within the 2-oxo-glutarate only transporters that is missing in general transporters. Cysteines which are functionally key residues are inferred to be mediating intra- or inter-reactive disulfide bond formation or using a thiol (sulfhydryl) group for transport or to be forming a metal binding site. When a disulfide bond prediction tool was employed, it showed with negligible doubt that the Cys-X-X Cys-X(19) -Cys region was a strong contender for 2 separate disulfide bonds, although the middle cysteine was predicted to be involved in both. In addition, Cluster 2 general Zea mays C4 transporters are shown to be more recalcitrant to mutations of cysteines, compared to Panicum and Oryza counterparts. The study of 2-oxoglutarate and its availability in the chloroplast could play a two-prong role in C4 plants: to be a candidate for synthesis of bundle sheath cell Rubisco enzyme, which makes up ~50% of plant proteins, via ammonia assimilation, and even playing a role in carbon-centered biochemical pathways. This study could greatly facilitate choices in the tinkering of the right transporters for a future C4 rice in a climate change impacted world.

Thioredoxins (TRXs) f and m are redox proteins that regulate key chloroplast processes. The existence of several isoforms of TRXs f and m indicates that these redox players have followed a specialization process throughout evolution. Current research efforts are focused on discerning the signalling role of the different TRX types and their isoforms in chloroplasts. Nonetheless, little is known about their function in non-photosynthetic plastids. For this purpose, we have carried out comprehensive expression analyses by using Arabidopsis thaliana TRXf (f1 and f2) and TRXm (m1, m2, m3 and m4) genes translationally fused to the green fluorescence protein (GFP). These analyses showed that TRX m has different localisation patterns inside chloroplasts, together with a putative dual subcellular localisation of TRX f1. Apart from mesophyll cells, these TRXs were also observed in reproductive organs, stomatal guard cells and roots. We also investigated whether photosynthesis, stomatal density and aperture or root structure were affected in the TRXs f and m loss-of-function Arabidopsis mutants. Remarkably, we immunodetected TRX m2 and the Calvin–Benson cycle fructose-1,6-bisphosphatase (cFBP1) in roots. After carrying out in vitro redox activation assays of cFBP1 by plastid TRXs, we propose that cFBP1 might be activated by TRX m2 in root plastids., This work has been funded by research projects BIO2009-07297 and BIO2015-65272 from the Spanish Ministry of Science and Innovation and the European Fund for Regional Development; project P07-CVI-2795 and BIO 154 from the Andalusian Regional Government, Spain; project BIO2012-33292 from the Spanish Ministry of Economy and Competitiveness; and by Grant PGC2018-096851-B-C21, from FEDER/Spanish Ministry of Science, Innovation and University: AEI, Spain.

Plant melanin is a dark polymerized polyphenolic substance that can by synthesized in seed tissues. Unlike well-defined enzymatic browning reaction leading to melanin synthesis in senescent and damaged plant tissues, melanin formation in intact tissues was not studied properly. Recently, melanin synthesis was demonstrated in chloroplast-derived melanoplasts in pericarp and husk cells of barley seeds. In barley, there are two independent genes, Blp1 and Alm1, affecting respectively the biosynthesis of melanin and chlorophyll in seeds. Even though different genetic systems are responsible for these traits, the localization of these biosynthetic pathways in the same organelle prompted us to conduct an in-depth study of the i:Bwalm1Blp1 line characterized by simultaneous chlorophyll deficiency caused by recessive allele alm1 and melanin accumulation controlled by dominant allele Blp1. This barley line and parental ones—Bowman, i:BwBlp1, and i:Bwalm1, which are characterized by different combinations of pigments chlorophyll and melanin in seeds—were subjected to a comparative cytological analysis. Three markers were analyzed: the presence of visible pigments, chlorophyll, and PsbA protein (a thylakoid membrane marker). Plastids of the barley pericarp and husk showed prominent differences among the lines, with internal structures that are more developed in husk cells. Although chlorophyll deficiency did not prevent melanogenesis in the spike of the hybrid line, a 7-day delay in melanization initiation and a decrease in its magnitude were observed in comparison with the melanin-and-chlorophyll–containing line. Thus, melanin biosynthesis is not related to photosynthetic processes directly but may be dependent on the presence of plastids with well-developed internal membranes.

Brassica napus L. (B. napus) is an important oil crop worldwide and it rapidly accumulates oil at late stage of seed maturation. However, little is known about the cellular mechanism of oil accumulation and seed color changes during the late stage of rapeseed development. Here, we analyzed the ultrastructure of seed coat, aleurone and cotyledon in embryos of B. napus from 25 to 70 days after flowering (DAF). The pigments, which were deposited on the cell wall of palisade cells in seed coat, determined dark black color of rapeseed. The chloroplasts degenerated into non-photosynthetic plastids which caused the green cotyledon to turn into yellow. The chloroplasts in aleurone and cotyledon cells respectively degenerated into remnants without inner and outer envelope membranes and ecoplasts with intact inner and outer envelope membranes. From 40 to 70 DAF, there were degraded chloroplasts without thylakoid, oil bodies contacting with plastids or protein bodies, big starch deposits of chloroplasts degrading into small particles then disappearing, and small endoplasmic reticulum (ER) in aleurone and cotyledon cells. Additionally, there were decreases of chlorophyll content and dramatic increases of oil content in rapeseed. These results suggested that the rapid oil accumulation was independent on the NADPH synthesized by photosynthesis of chloroplasts and probably utilized other sources of reductant, such as the oxidative pentose phosphate pathway during the late stage of rapeseed development. The triacylglycerol assembly presumably utilizes the enzymes in the plastid, cytosol or oil body of cotyledon and aleurone cells.

Twelve-oxo-phytodienoic acid (OPDA), the cyclopentenone precursor of jasmonic acid (JA), is required for the wounding response of plants. OPDA is derived from plastid-localized α-linolenic acid (α-LeA; 18:3) via the octadecanoid pathway, and is further exported from plastids to the cytosol for JA biosynthesis. However, the mechanism of OPDA transport from plastids has yet to be elucidated. In the current study, a plastid inner envelope-localized protein, designated 12-oxo-Phtyodienoic Acid Transporter 1 (OPDAT1), was identified and shown to potentially be involved in OPDA export from plastids, in Populus trichocarpa. Torr. OPDAT1 is expressed predominantly in young leaves of P. trichocarpa. Functional expression of OPDAT1 in yeast cells revealed that OPDAT1 is involved in OPDA transport. Loss-of-function of OPDAT1 in poplar resulted in increased accumulation of OPDA in the extracted plastids and a reduction in JA concentration, whereas an OPDAT1-overexpressing line showed a reverse tendency in OPDA accumulation and JA biosynthesis. OPDAT1 transcripts were rapidly induced by mechanical wounding of leaves, and an opdat1 mutant transgenic plant displayed increased susceptibility to spider mite (Tetranychus urticae) infestation. Collectively, these data suggest that OPDAT1 is an inner envelope transporter for OPDA, and this has potential implications for JA biosynthesis in poplar under environmental stresses.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway., Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms., The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued., Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform., This work was funded by the European Regional Development Fund (FEDER) and the Spanish Agencia Estatal de Investigación (grants BIO2017-84041-P and BIO2017-90877-REDT) and Generalitat de Catalunya (2017SGR-710) to MRC. Support by the collaborative European Union’s Horizon 2020 (EU-H2020) ERA-IB-2 (Industrial Biotechnology) BioProMo project to MRC (PCIN-2015-103), RK and JB (053-80-725) is also acknowledged. CRAG is financially supported by the Severo Ochoa Programme for Centres of Excellence in R&D 2016–2019 (SEV-2015-0533) and the Generalitat de Catalunya CERCA Programme. MVB was funded with a Spanish Ministry of Education, Culture and Sports PhD fellowship (FPU14/05142) and a EU-H2020 COST Action CA15136 (EuroCaroten) short-stay fellowship. ME is supported by a Spanish Agencia Estatal de Investigación (BES-2017-080652) PhD fellowship. IFS is supported by the EU-H2020 Marie S. Curie Action 753301 (Arcatom).

Key message Macroscopic, ultrastructural, and molecular features—like a ball shape, the presence of starch granules, and the up-regulation of genes involved in carbohydrate metabolism and secondary metabolite biosynthesis—distinguish PT regions within a callus. Abstract The modification of the mass of pluripotent cells into de novo shoot bud regeneration is highly relevant to developmental biology and for agriculture and biotechnology. This study deals with protuberances (PT), structures that appear during the organogenic long-term culturing of callus (OC) in kiwifruit. These ball-shaped regions of callus might be considered the first morphological sign of the subsequent shoot bud development. Sections of PT show the regular arrangement of some cells, especially on the surface, in contrast to the regions of OC beyond the PT. The cells of OC possess chloroplasts; however, starch granules were observed only in PTs’ plastids. Transcriptomic data revealed unique gene expression for each kind of sample: OC, PT, and PT with visible shoot buds (PT–SH). Higher expression of the gene involved in lipid (glycerol-3-phosphate acyltransferase 5 [GPAT5]), carbohydrate (granule-bound starch synthase 1 [GBSS1]), and secondary metabolite (beta-glucosidase 45 [BGL45]) pathways were detected in PT and could be proposed as the markers of these structures. The up-regulation of the regulatory associated protein of TOR (RAPTOR1) was found in PT–SH. The highest expression of the actinidain gene in leaves from two-year-old regenerated plants suggests that the synthesis of this protein takes place in fully developed organs. The findings indicate that PT and PT–SH are specific structures within OC but have more features in common with callus tissue than with organs.

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell-pepper fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b6f complex, the ATPase complex as well as Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids such as the oxidative pentose phosphate pathway (OPPP), amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins declines but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation may be consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper, that lack a respiratory burst at the onset of fruit ripening.

Diatoms are a diverse group of photosynthetic unicellular algae with a plastid of red-algal origin. As prolific primary producers in the ocean, diatoms fix as much carbon as all rainforests combined. The molecular mechanisms that contribute to the high photosynthetic productivity and ecological success of diatoms are however not yet fully understood. Using the model diatom Phaeodactylum tricornutum, here we show rhythmic transcript accumulation of plastid psaA, psbA, petB, and atpB genes as driven by a free running circadian clock. Treatment with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea overrides the circadian signal by markedly downregulating transcription of psaA, petB, and atpB genes but not the psbA gene. Changes in light quantity produce little change in plastid gene transcription while the effect of light quality seems modest with only the psaA gene responding in a pattern that is dependent on the redox state of the plastoquinone pool. The significance of these plastid transcriptional responses and the identity of the underlying genetic control systems are discussed with relevance to diatom photosynthetic acclimation.

Summary Carotenoids are isoprenoid compounds synthesized by all photosynthetic and some non‐photosynthetic organisms. They are essential for photosynthesis and contribute to many other aspects of a plant's life. The oxidative breakdown of carotenoids gives rise to the formation of a diverse family of essential metabolites called apocarotenoids. This metabolic process either takes place spontaneously through reactive oxygen species or is catalyzed by enzymes generally belonging to the CAROTENOID CLEAVAGE DIOXYGENASE family. Apocarotenoids include the phytohormones abscisic acid and strigolactones (SLs), signaling molecules and growth regulators. Abscisic acid and SLs are vital in regulating plant growth, development and stress response. SLs are also an essential component in plants’ rhizospheric communication with symbionts and parasites. Other apocarotenoid small molecules, such as blumenols, mycorradicins, zaxinone, anchorene, β‐cyclocitral, β‐cyclogeranic acid, β‐ionone and loliolide, are involved in plant growth and development, and/or contribute to different processes, including arbuscular mycorrhiza symbiosis, abiotic stress response, plant–plant and plant–herbivore interactions and plastid retrograde signaling. There are also indications for the presence of structurally unidentified linear cis‐carotene‐derived apocarotenoids, which are presumed to modulate plastid biogenesis and leaf morphology, among other developmental processes. Here, we provide an overview on the biology of old, recently discovered and supposed plant apocarotenoid signaling molecules, describing their biosynthesis, developmental and physiological functions, and role as a messenger in plant communication., Significance Statement Apocarotenoids are carotenoid‐derived small molecules with regulatory functions, including cellular signaling, growth regulation and communication with surrounding organisms. Owing to these functions, apocarotenoids can influence plant physiology, development and plant–plant/plant–microorganisms interactions, making them a major focus of study in agriculture and fundamental research.

The valuable terpenoids, such as artemisinin acid, have achieved bioproduction in the chassis of microbes recently. In this study, Marchantia paleacea L, a promising plant synthetic biology chassis, was used to explore the possibility of patchoulol production by constructing a synthetic biology pathway composed of FPS and PTS. The experiment results show that the maximum yields based on the cytoplasm and plastid pathway were 621.56 and 1006.45 μg/g, respectively. However, there is no statistically significant difference in the yield of patchoulol between transformant plants with different subcellular compartment-targeting pathways. However, it was found that the highest yield of patchoulol was achieved in transformant plants with similar transcription levels of FPS and PTS. Also, the optimized transcription ratio between PTS and FPS is determined at 1.12 based on statistical analysis and model simulation. Therefore, two kinds of new optimized pathway vectors were constructed. One is based on the fusion protein method, and the other is based on protein expression individually, in which the same promoter and terminator were used to derive the expression of both FPS and PTS. The effect of pathway optimization was tested by transient and stable transformation. The production of patchoulol in transient transformation was the same for the two abovementioned kinds of matching pathway and higher than that for the original pathway. Also, in stable transformation, the yield of patchoulol reached up to 3250.30 μg/g, being three times the maximum content before optimization. It is suggested that M. paleacea is a powerful plant chassis for terpenoid synthetic biology and the matching between enzymes may be the key factor in determining the metabolic flux of the pathway in the study of synthetic biology.

Plastids are specialized organelles found in plants, which are endowed with their own genomes, and differ in many respects from the intracellular compartments of organisms belonging to other kingdoms of life. They differentiate into diverse, plant organ-specific variants, and are perhaps the most versatile organelles known. Chloroplasts are the green plastids in the leaves and stems of plants, whose primary function is photosynthesis. In response to environmental changes, chloroplasts use several mechanisms to coordinate their photosynthetic activities with nuclear gene expression and other metabolic pathways. Here, we focus on a redox-based regulatory network composed of thioredoxins (TRX) and TRX-like proteins. Among multiple redox-controlled metabolic activities in chloroplasts, tetrapyrrole biosynthesis is particularly rich in TRX-dependent enzymes. This review summarizes the effects of plastid-localized reductants on several enzymes of this pathway, which have been shown to undergo dithiol-disulfide transitions. We describe the impact of TRX-dependent control on the activity, stability and interactions of these enzymes, and assess its contribution to the provision of adequate supplies of metabolic intermediates in the face of diurnal and more rapid and transient changes in light levels and other environmental factors.

p-Menthanes are among the most valuable groups of monoterpenoids produced by plants. Products such as menthol, carvone, pulegone, and limonene are well-known fragrance compounds that are also used as pesticides or medicines. In flowering plants, they are synthesized and stored in specialized anatomical structures such as glandular trichomes (GT), secretory cavities, and root resin ducts. These metabolites originate through the 2C-methyl-d-erythritol-4-phosphate precursor pathway in the plastids of GT secretory cells but also require the activity of prenyltransferases and modifying enzymes in the mitochondria and endoplasmic reticulum to complete their functionalization to biologically active forms. (−)-Menthol is the best characterized of these and is pharmacologically active due to its affinity for the Cold and Menthol Receptor 1 and the κ-Opioid Receptor. p-Menthanes are found throughout the plant kingdom, and although their essential oils are still obtained mostly through traditional agriculture, synthetic biology approaches increasingly enable large scale production of economically valuable examples in microbes and cell free systems. Limonene is the model p-menthane monoterpene, and detailed studies of (+)-limonene synthase and (−)-limonene synthase crystal structures have enabled identification of catalytic residues and determinants of stereospecificity, which has implications for the ecological properties of these volatile products in defense and interspecific signaling. This review examines the chemical diversity of the p-menthanes in the plant kingdom, their biosynthetic origins in specialized anatomical structures, and the enzymology of p-menthane biosynthetic enzymes from diverse plant families.

Euglena gracilis is a photosynthetic flagellate possessing chlorophyte-derived secondary plastids that are enclosed by only three enveloping membranes, unlike most secondary plastids, which are surrounded by four membranes. It has generally been assumed that the two innermost E. gracilis plastid envelopes originated from the primary plastid, while the outermost is of eukaryotic origin. It was suggested that nucleus-encoded plastid proteins pass through the middle and innermost plastid envelopes of E. gracilis by machinery homologous to the translocons of outer and inner chloroplast membranes, respectively. Although recent genomic, transcriptomic, and proteomic data proved the presence of a reduced form of the translocon of inner membrane, they failed to identify any outer-membrane translocon homologs, which raised the question of the origin of E. gracilis's middle plastid envelope. Here, we compared the lipid composition of whole cells of the pigmented E. gracilis strain Z and two bleached mutants that lack detectable plastid structures, W10BSmL and WgmZOflL We determined the lipid composition of E. gracilis strain Z mitochondria and plastids, and of plastid subfractions (thylakoids and envelopes), using HPLC high-resolution tandem mass spectrometry, thin-layer chromatography, and gas chromatography-flame ionization detection analytical techniques. Phosphoglycerolipids are the main structural lipids in mitochondria, while glycosyldiacylglycerols are the major structural lipids of plastids and also predominate in extracts of whole mixotrophic cells. Glycosyldiacylglycerols were detected in both bleached mutants, indicating that mutant cells retain some plastid remnants. Additionally, we discuss the origin of the E. gracilis middle plastid envelope based on the lipid composition of envelope fraction.

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

Background Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. Results With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. Conclusions Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

Chlamydomonas reinhardtii P.A. Dangeard is a unicellular green alga capable to assimilate acetate. C. reinhardtii growth and metabolism distinctly depend on trophic conditions. Its influence on batch culture interferes with changes in the medium composition and metabolism of microalgae occurring during culture growth. The aim of this work was to estimate the effect of acetate on changes in the expression of 32 genes encoding enzymes of central metabolism and plastid transporters during growth of batch cultures of C. reinhardtii cc-124. In autotrophic conditions, transcription profiles considerably differed from the profiles in the presence of acetate. The strongest influence of trophic conditions was observed in the log phase of growth. In the presence of acetate, a more intense expression of gene ACS2 encoding plastid acetyl-CoA synthase and of the genes encoding subunits of enzymes directing acetyl groups to the synthesis of fatty acids was recorded. Elevated expression of genes PCK1 and PPT1 under mixotrophic conditions may be corresponded to the entry of acetate carbon to gluconeogenesis. In the presence of acetate, a high expression level of the starch metabolism genes was observed. Autotrophic conditions were notable for an elevated accumulation of transcripts of the genes encoding subunits of citrate lyase, which may be responsible for an outflow of acetyl groups from the Krebs cycle. Moreover, a higher level of expression was shown for genes of plastid transporters participating in the export of sugars from plastids, which is associated with the redistribution of reducing power in the cell. It was concluded that, on the level of transcription, similarity between mixotrophic and autotrophic cultures becomes more pronounced in the course of development.

Chlorophyll biosynthesis and chloroplast development are crucial to photosynthesis and plant growth, but their regulatory mechanism remains elusive in many crop species. We isolated a Brassica napus yellow-virescent leaf (yvl) mutant, which exhibited yellow-younger-leaf and virescent-older-leaf with decreased chlorophyll accumulation and delayed chloroplast development. We mapped yvl locus to a 70-kb interval between molecular markers yvl-O10 and InDel-O6 on chromosome A03 in BC2F2 population using whole genome re-sequencing and bulked segregant analysis. The mutant had a ‘C’ to ‘T’ substitution in the coding sequence of BnaA03.CHLH, which encodes putative H subunit of Mg-protoporphyrin IX chelatase (CHLH). The mutation resulted in an imperfect protein structure and reduced activity of CHLH. It also hampered the plastid encoded RNA polymerase which transcribes regulatory genes of photosystem II and I. Consequently, the chlorophyll a/b and carotenoid contents were reduced and the chloroplast ultrastructure was degraded in yvl mutant. These results explain that a single nucleotide mutation in BnaA03.CHLH impairs PEP activity to disrupt chloroplast development and chlorophyll biosynthesis in B. napus.

The aim of the present study was to investigate the photosynthetic properties and transcriptomic profiles of wildtype and chlorophyll (Chl) b-lacking rice (Oryza sativa L.). The plastid ultrastructure of the Chl b-lacking rice (i.e., loss of starch granules, abundant vesicles, and abundant plastoglobuli) indicated abnormal plastid development, whereas the analysis of transcriptome profiles and differentially expressed genes revealed that gene encoding PsbR (PSII core protein) was downregulated in the mutant, thereby reducing the Chl accumulation of the mutant. Meanwhile, in regards to Chl biosynthesis and degradation pathways, GluTR gene was downregulated, whereas UROD, CPOX, and MgCH genes were upregulated. The qPCR results were generally consistent with those of the transcription analysis, except for the finding that NOL genes, which regulate Chl b degradation, were upregulated. These results suggest that both the reduction in Chl accumulation and increase in conversion rate of Chl b to Chl a caused Chl a/b ratio amplification in mutant. The present study also provides evidence for Chl b degradation via pheophorbide b.

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans., Complex polyketides are usually produced by microbes, whereas the origin of polyketides found in animals remained unknown. This study shows that sacoglossan animals, such as sea slugs, employ fatty acid synthase-like proteins to produce microbe-like polyketides.

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process. © 2020 American Society of Plant Biologists. ISSN:1040-4651 ISSN:1531-298X ISSN:1532-298X

Significance Carotenoids are essential molecules in oxygenic photoautotrophs, and they fulfill essential requirements for human and animal nutrition. How carotenoid accumulation is regulated in the chloroplast, a cyanobacterium-derived organelle, remains poorly understood, despite significant advancements in identifying enzymes of the carotenoid biosynthetic pathway. This study identifies a role of chloroplast Sec14-like 1 (CPSFL1), a CRAL-TRIO protein of eukaryotic origin, in modulation of carotenoid biosynthesis and accumulation in the chloroplast. The CPSFL1 protein represents an isoprenoid- and carotenoid-binding protein that associates with membranes through interactions with the phospholipid phosphatidic acid. These findings have implications for understanding carotenoid biosynthesis and optimizing algal carotenoid nutritional quality., Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of Chlamydomonas reinhardtii and isolated cpsfl1. The cpsfl1 mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The CPSFL1 gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The cpsfl1 mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and β-carotene when expressed in Escherichia coli and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

Summary The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of n ifU and n ifS co‐expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis (Arabidopsis thaliana) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO(2) atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries.

The balance between cellular carbon (C) and nitrogen (N) must be tightly coordinated to sustain optimal growth and development in plants. In chloroplasts, photosynthesis converts inorganic C to organic C, which is important for maintenance of C content in plant cells. However, little is known about the role of chloroplasts in C/N balance. Here, we identified a nuclear‐encoded protein LOW PHOTOSYNTHETIC EFFICIENCY2 (LPE2) that it is required for photosynthesis and C/N balance in Arabidopsis. LPE2 is specifically localized in the chloroplast. Both loss‐of‐function mutants, lpe2‐1 and lpe2‐2, showed lower photosynthetic activity, characterized by slower electron transport and lower PSII quantum yield than the wild type. Notably, LPE2 is predicted to encode the plastid ribosomal protein S21 (RPS21). Deficiency of LPE2 significantly perturbed the thylakoid membrane composition and plastid protein accumulation, although the transcription of plastid genes is not affected obviously. More interestingly, transcriptome analysis indicated that the loss of LPE2 altered the expression of C and N response related genes in nucleus, which is confirmed by quantitative real‐time‐polymerase chain reaction. Moreover, deficiency of LPE2 suppressed the response of C/N balance in physiological level. Taken together, our findings suggest that LPE2 plays dual roles in photosynthesis and the response to C/N balance., We demonstrate that plastid ribosomal protein LPE2 plays dual roles in photosynthesis and the response to C/N balance, and reveal that the putative plastid translation signal mediated by LPE2 regulates response to C/N balance probably via retrograde signaling.

Triacylglycerols (TAGs) are the major storage form of seed oil in oilseed plants. They are biosynthesized de novo in seed plastids and then transported into the endoplasmic reticulum. However, the transport mechanism for plastid fatty acids in developing seeds remains unknown. Here, we isolated two novel plastid fatty acid exporters (FATTYACID EXPORT 2 [FAX2] and FAX4, respectively) specifically abundant in seed embryos during the seed-filling stage in Arabidopsis (Arabidopsis thaliana). FAX2 and FAX4 were both localized to the chloroplast membrane. FAX2 and FAX4 loss-of-function mutations caused deficiencies in embryo and cotyledon development. Seeds of fax2fax4 double mutants exhibited significantly reduced TAG contents but elevated levels of plastid lipid contents compared with those of wild-type plants. By contrast, overexpression of FAX2 or FAX4 enhanced TAG deposition. Seed-feeding experiments showed that the two FAX proteins transported (14)C-plastid fatty acids and (13)C-oleic acids for TAG biosynthesis during the seed-filling stage. Together, our data demonstrate that FAX2 and FAX4 play critical roles in transporting plastid fatty acids for TAG biosynthesis during seed embryo development. These two transporters may have broad application for increasing oil yield in oilseed crops.

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far‐reaching ecological consequences. Recently, 2‐alkyl‐4‐quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom–bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half‐maximal inhibitory concentrations reaching the sub‐micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2‐heptyl‐4‐quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b 6 f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments., Double trouble: In the light, 2‐heptyl‐4‐quinolone (HHQ) primarily blocks photosynthetic electron flow at the cytochrome b 6 f complex in the diatom P. tricornutum. In darkness, HHQ inhibits mitochondrial respiration, resulting in a decreased ATP supply, which ultimately leads to a decreased electric field strength in the thylakoid membranes inside the chloroplast of diatoms.

Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals. The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mutants, while the physiological role of their residual plastid DNAs remains unclear. In this study, we obtained five bleached mutants by ofloxacin (Ofl) treatment, identified 12 residual plastid genes in five bleached mutants, and determined the mRNA levels in the wild type E. gracilis (WT) and one bleached mutant (OflB2) under dark and light stimulation conditions by quantitative reverse transcribed PCR (qRT-PCR). Results show that the expression of all selected plastid genes in both WT and OflB2 mutant did not change significantly in darkness, while their responses to light stimulation were different. Under the light stimulation conditions, half of the genes did not change significantly, while most of the other genes were down-regulated in OflB2 mutant and up-regulated in WT. Therefore, the bleached mutant retains part of the plastid genome and the plastid relic is responsive to light. Our research will help to understand the functions of residual plastid DNA and evolution of chloroplasts.

Chloroplasts are photosynthetic plant organelles descended from a bacterial ancestor. The vast majority of chloroplast proteins are synthesized in the cytosol and then imported into the chloroplast post-translationally. Translocation complexes exist in the organelle's outer and inner envelope membranes (termed TOC and TIC, respectively) to facilitate protein import. These systems recognize chloroplast precursor proteins and mediate their import in an energy-dependent manner. However, many unanswered questions remain regarding mechanistic details of the import process and the participation and functions of individual components; for example, the cytosolic events that mediate protein delivery to chloroplasts, the composition of the TIC apparatus, and the nature of the protein import motor all require resolution. The flux of proteins through TOC and TIC varies greatly throughout development and in response to specific environmental cues. The import process is, therefore, tightly regulated, and it has emerged that the ubiquitin-proteasome system (UPS) plays a key role in this regard, acting at several different steps in the process. The UPS is involved in: the selective degradation of transcription factors that co-ordinate the expression of chloroplast precursor proteins; the removal of unimported chloroplast precursor proteins in the cytosol; the inhibition of chloroplast biogenesis pre-germination; and the reconfiguration of the TOC apparatus in response to developmental and environmental signals in a process termed chloroplast-associated protein degradation. In this review, we highlight recent advances in our understanding of protein import into chloroplasts and how this process is regulated by the UPS.

International audience; Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O$_2$. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O$_2$ photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O$_2$-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K+ transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch synthesis in seeds, tubers and fruits. UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of sucrose metabolism in the cytosol while alkaline phosphatase (ALP) is a marker enzyme of the amyloplast that keeps the production of ADPG by removing PPi. Unripe banana accumulates starch in the pulp during development, while ripe fruits are characterized by the accumulation of soluble sugars. The aim of the study was to compare starch granule structure, carbohydrate levels, subcellular location and activities of three enzymes: AGPase, UGPase and ALP. Protein extracts from the cytosolic and amyloplastidial fractions were obtained from the pulp of banana fruit at three developmental stages (11, 16 and 21 weeks after flowering) and analyzed by electrophoresis and immunodetection. Protein profiles were similar during ripening, showing a main electrophoretic band at 50-55 kDa. Higher protein content was found in the cytosolic than in the amyloplastidial fraction. Starch granules and ALP activity were enriched in the amyloplast, whereas AGPase showed a subcellular distribution similar to UGPase. Immunoblot analysis also confirmed the presence of AGPase in both cytosol and amyloplast. AGPase activity was higher in the cytosol than in the amyloplast. Both AGPase activity and western blot band intensity were highest at 16 weeks. UGPase activity was highest at 21 weeks. We conclude that cytosolic production of ADP-glucose is not an exclusive feature of cereal endosperms due to plant breeding, but it also occurs in fruits of non-domesticated plants such as tropical banana (Musa acuminata). This work increases our understanding about pyrophosphorylase activities in the pulp of banana fruit.

Arabidopsis (Arabidopsis thaliana), like most dicotyledonous plants, expresses a multicomponent, heteromeric acetyl-CoA carboxylase (htACCase), which catalyzes the generation of the malonyl-CoA precursor of de novo fatty acid biosynthesis. This enzyme consists of four catalytic subunits: biotin carboxylase (BC), carboxyltransferase (CT)-α, CT-β, and biotin carboxyl carrier protein (BCCP1 or BCCP2). By coexpressing combinations of components in a bacterial expression system, we demonstrate noncatalytic BADCs facilitate the assembly and activation of BCCP–BADC–BC subcomplexes catalyzing the bicarbonate-dependent hydrolysis of ATP, which is the first half-reaction catalyzed by the htACCase enzyme. Although BADC proteins do not directly impact formation of the CT–αβ subcomplex, the BADC-facilitated BCCP–BADC–BC subcomplex can more readily interact with the CT–αβ subcomplex to facilitate the generation of malonyl-CoA. The Arabidopsis genome encodes three BADC isoforms (BADC1, BADC2, and BADC3), and BADC2 and BADC3 (rather than BADC1), in combination with BCCP1, best support this quaternary-structural organization and catalytic activation of the htACCase enzyme. Physiological genetic studies validate these attributes as Arabidopsis double mutants singularly expressing BADC2, BADC3, or BADC1 present increasingly greater deleterious impacts on morphological and biochemical phenotypes. Specifically, plants expressing only BADC2 develop normally, plants only expressing BADC3 suffer a stunted root-growth phenotype, and plants expressing only BADC1 are embryo-lethal. The latter phenotype may also be associated with the distinct suborganelle localization of BADC1 in plastids as compared to the localization of the other two BADC homologs. These finding can inspire novel strategies to improve the biological sources of fats and oils for dietary and industrial applications.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium) collected in surface seawaters. We showed that this symbiosis is a very dynamic system whereby symbionts interact with different host cells via extracellular vesicles within the “greenhouse-like” colony. 3D electron microscopy revealed that the volume of the photosynthetic apparatus (plastid and pyrenoid) of the microalgae increased in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were assimilated and stored in the symbiotic microalga in starch granules and purine crystals, respectively. Nitrogen was also allocated to the algal nucleus (nucleolus). After 3 hours, low13C and15N transfer was detected in the host Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (ca 40 ppm) and two-fold higher in the host, whereas copper concentration increased in symbiotic microalgae (up to 6900 ppm) and was detected in the host cell and extracellular vesicles. Sulfur mapping also pinpointed the importance of this nutrient for the algal metabolism. This study, which revealed subcellular changes of the morphology and nutrient homeostasis in symbiotic microalgae, improves our understanding on the metabolism of this widespread and abundant oceanic symbiosis and paves the way for more studies to investigate the metabolites exchanged.

LEA proteins are involved in plant stress tolerance. In Arabidopsis, the LEA_4 Pfam group is the biggest group with the majority of its members being expressed in dry seeds. To assess subcellular localization in vivo, we investigated 11 seed-expressed LEA_4 proteins in embryos dissected from dry seeds expressing LEA_4 fusion proteins under its native promoters with the Venus fluorescent protein (proLEA_4::LEA_4:Venus). LEA_4 proteins were shown to be localized in the endoplasmic reticulum, nucleus, mitochondria, and plastids. LEA9, in addition to the nucleus, was also found in cytoplasmic condensates in dry seeds dependent on cellular hydration level. Most investigated LEA_4 proteins were detected in 4-d-old seedlings. In addition, we assessed bioinformatic tools for predicting subcellular localization and promoter motifs of 11 seed-expressed LEA_4 proteins. Ratiometric bimolecular fluorescence complementation assays showed that LEA7, LEA29, and LEA48 form homodimers while heterodimers were formed between LEA7-LEA29 and LEA42-LEA48 in tobacco leaves. Interestingly, LEA48 homodimers and LEA42-LEA48 heterodimers formed droplets structures with liquid-like behavior. These structures, along with LEA9 cytoplasmic condensates, may have been formed through liquid-liquid phase separation. These findings suggest possible important roles of LLPS for LEA protein functions.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention., Author summary Toxoplasma gondii is a ubiquitous unicellular parasite that harbours two organelles of endosymbiotic origin: the mitochondrion, and a relict plastid named the apicoplast. Each one of these organelles contains its own machinery for synthesizing iron-sulfur clusters, which are important protein co-factors. In this study, we show that interfering with either the mitochondrial or the plastidic iron-sulfur cluster synthesizing machinery has a profound impact on parasite growth. However, while disrupting the plastidic pathway led to an irreversible loss of the organelle and subsequent death of the parasite, disrupting the mitochondrial pathway led to conversion of the parasites into a stress resistance form. We used a comparative quantitative proteomic analysis of the mutants, combined with experimental validation, to provide mechanistic clues into these different phenotypic outcomes. Although the consequences of disrupting each pathway were manifold, our data highlighted potential changes at the metabolic level. For instance, the plastidic iron-sulfur cluster synthesis pathway may be important for maintaining the lipid homeostasis of the parasites, while the mitochondrial pathway is likely crucial for maintaining their respiratory capacity. Interestingly, we have discovered that other mutants severely impacted for mitochondrial function, in particular the respiratory chain, are able to survive and initiate conversion to the stress resistance form. This illustrates a different capacity for T. gondii to adapt for survival in response to distinct metabolic dysregulations.

Apicomplexan parasites, such as Toxoplasma gondii, are unusual in that each cell contains a single apicoplast, a plastid-like organelle that compartmentalizes enzymes involved in the essential 2C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. The last two enzymatic steps in this organellar pathway require electrons from a redox carrier. However, the small iron-sulfur cluster-containing protein ferredoxin, a likely candidate for this function, has not been investigated in this context. We show here that inducible knockdown of T. gondii ferredoxin results in progressive inhibition of growth and eventual parasite death. Surprisingly, this phenotype is not accompanied by ultrastructural changes in the apicoplast or overall cell morphology. The knockdown of ferredoxin was instead associated with a dramatic decrease in cellular levels of the last two metabolites in isoprenoid biosynthesis, 1-hydroxy-2-methyl-2-(E)- butenyl-4-pyrophosphate, and isomeric dimethylallyl pyrophosphate/isopentenyl pyrophosphate. Ferredoxin depletion was also observed to impair gliding motility, consistent with isoprenoid metabolites being important for dolichol biosynthesis, protein prenylation, and modification of other proteins involved in motility. Significantly, pharmacological inhibition of isoprenoid synthesis of the host cell exacerbated the impact of ferredoxin depletion on parasite replication, suggesting that the slow onset of parasite death after ferredoxin depletion is because of isoprenoid scavenging from the host cell and leading to partial compensation of the depleted parasite metabolites upon ferredoxin knockdown. Overall, these findings show that ferredoxin has an essential physiological function as an electron donor for the 2C-methyl-D-erythritol 4-phosphate pathway and is a potential drug target for apicomplexan parasites.

Starch accumulates in the plastids of green plant tissue during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the β-amylase (BAM) family, which in Arabidopsis thaliana (At), includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized β-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since BAM2 is found in more ancient lineages, it was hypothesized that BAM7 evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to have a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. We hypothesize that these genes may encode a BAM2-like protein although they are currently annotated as BAM7-like genes. To test this hypothesis, we designed a cDNA of the short form of corn BAM7 (ZmBAM7-S) for expression in E. coli. Small Angle X-Ray Scattering data indicate that ZmBAM7-S has a tetrameric solution structure more similar to that of AtBAM2 than AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding β-amylase gene structure could have impacts on the current annotation of genes.

This research was funded by the Scientific and Technological Research Council of Turkey (TUBITAK), grant number KBAG-115Z470 to E.G. The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5-beta-reductases (P5 beta R1, P5 beta R2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3 beta-HSD, P5 beta R1 and P5 beta R2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3 beta-HSD and P5 beta R1 are cytoplasmic, and P5 beta R2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3 beta-HSD, P5 beta R1 and P5 beta R2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering. Scientific and Technological Research Council of Turkey (TUBITAK)

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution., Author summary We simulated the evolution of four photosynthesis genes that are coded by the chloroplast genome of flowering plants in order to investigate the role of natural selection. In particular we were interested in whether or not selection can influence the evolution of features that do not affect the protein coded by the gene but that do affect the expression of the gene. We developed a model of mutation and then assume that there is no selection in simulations to generate expected patterns of evolution. Importantly, our mutation model accounts for a complex feature of chloroplast DNA which is that nucleotides near the site mutating affect the type of mutation that occurs. We found that one gene in particular has not evolved as predicted by our simulations but, rather, has evolved in a manner that suggests that mutations which affect the level of gene expression have been under natural selection.

Veterinary antibiotics, including erythromycin (Ery) and chlortetracycline (CTC), are often detected in agricultural land. Although these contaminants affect plant growth and development, their effects on crops remain elusive. In this study, the effects of Ery and CTC on plant growth were investigated and compared by analyzing transcript abundance in Brassica campestris seedlings. Treatment with Ery and/or CTC reduced chlorophyll content in leaves and photosynthetic efficiency. Examination of the chloroplast ultrastructure revealed the presence of abnormally shaped plastids in response to Ery and CTC treatments. The antibiotics produced similar phenotypes of lower accumulation of photosynthetic genes, including RBCL and LHCB1.1. Analysis of the transcript levels revealed that Ery and CTC differentially down-regulated genes involved in the tetrapyrrole biosynthetic pathway and primary root growth. In the presence of Ery and CTC, chloroplasts were undeveloped and photosynthesis efficiency was reduced. These results suggest that both Ery and CTC individually affect gene expression and influence plant physiological activity, independently of one another.

SummaryOne of the main unsolved questions regarding photosynthetic sea slugs is how the slug plastids handle photoinhibition of Photosystem II. Photoinhibition has not been studied in detail in these animals although resilience against photoinhibition might obviously explain the longevity of plastids inside animal cytosol.Light response and action spectrum of photoinhibition were measured from the slug Elysia timida and its prey alga Acetabularia acetabulum. Plastid packing in the slugs and algae was compared with spectroscopic and microscopic methods. The importance of plastid concentration was also estimated by measuring photoinhibition from starved slugs.Compared to A. acetabulum, E. timida is highly resistant against photoinhibition. The resilience of the slugs is even more pronounced in the UV-region, as the slug tissue screens UV radiation. The plastids in the slug tissue are tightly packed, and the outer plastids protect the inner ones from photoinhibition.The sea slug E. timida protects its plastids from photoinhibition by screening UV radiation and packing the plastids tightly in its tissues. Both mechanisms enhance the longevity of the plastids in slug cytosol and ameliorate the need for repair of photoinhibited Photosystem II.

Three strains of Euglena mutabilis were isolated from sediments in acidic inland water systems (pH = 3.4–4.7), in Southwestern Japan—acid mine drainage in Sensui (Fukuoka), cold sulfidic spring in Bougatsuru (Oita), and a temporal pool in the Ebinokogen volcanic area (Miyazaki). All strains grew well in acidic media at pH 3.07. Phylogenetic analysis among these three strains showed high similarities to plastid SSU and nuclear SSU rRNA gene sequences (99.86% and 99.76%, respectively). They were closely related to the cultured isolates from other highly acidic habitats (pH = 2.0–5.9). Concentration of sulfate, aluminum, calcium, and iron had 7–70 fold of differences among the three studied habitats. Our results imply that the rRNA genes of E. mutabilis have compensated for their low genetic diversity by adapting to a wide pH range, as well as various water chemistry of habitats.

Autoluminescent plants that express a bacterial bioluminescence gene cluster1 have not been widely adopted due to requisite expression in plastids and low light output. Alternatively, we have engineered tobacco lines expressing a fungal bioluminescent system2, which converts caffeic acid present in all plants into luciferin, and report self-sustained luminescence easily visible to the naked eye. Our findings might underpin development of a suite of imaging tools for plants.