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Herbivore-induced plant volatiles (HIPVs) play an important role in insect resistance. As a common HIPV in tea plants (Camellia sinensis), β-ocimene has shown anti-insect function in other plants. However, whether β-ocimene in tea plants also provides insect resistance, and its mechanism of synthesis and emission are unknown. In this study, β-ocimene was confirmed to interfere with tea geometrid growth via signaling. Light was identified as the key factor controlling regular emission of β-ocimene induced by the wounding from tea geometrids. β-Ocimene synthase (CsBOS1) was located in plastids and catalyzed β-ocimene formation in overexpressed tobacco. CsBOS1 expression in tea leaves attacked by tea geometrids showed a day-low and night-high variation pattern, while CsABCG expression involved in volatile emission showed the opposite pattern. These two genes might regulate the regular β-ocimene emission from tea plants induced by tea geometrid attack. This study advances the understanding on HIPV emission and signaling in tea plants.

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.

Arabidopsis POLLUX -like homologs PEC1 and PEC2 represent plastid envelope membrane ion channels with K+ conductivity that are required for the stress-triggered Ca2+ release into the stroma., Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

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.

In egy1 (ethylene-dependent gravitropism-deficient and yellow-green 1) mutants of Arabidopsis thaliana (L.) Heynh., there is reduced granal thylakoids, smaller size and less number of plastids in endodermal cells, suggesting that EGY1 protein is required for chloroplast development. Chloroplast is the apparatus of photosynthesis, how do egy1 mutants affect photosynthesis? Here we have further characterized the function of EGY1 protein in photosynthesis. The chlorophyll content and Fv/Fm (maximum photochemical efficiency of PSII) decreased much more rapidly with increasing leaf age in egy1 mutants than in wild-type (WT) plants of A. thaliana. The results of blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BN-SDS-PAGE) suggested that EGY1 was associated with photosystem II (PSII) supercomplexes. The spectroscopic and immunoblot analyses further showed that accelerated reductions in the abundance of D1, D2 and light harvesting complex II (LHCII) occurred with increasing leaf age in egy1 mutants. Moreover, PSII proteins (D1 and LHCII) were less stable in egy1 mutants than in WT plants under high light condition. Pull-down assays showed that EGY1 was colocalized with D2 protein. These results showed that loss-function of EGY1 results in accelerated reduction of PSII and in decreased stability of PSII, suggesting that defection of EGY1 results in impaired photosynthesis through breaking down the interaction of EGY1 with PSII in A. thaliana.

Plastids are involved in phytohormone metabolism as well as photosynthesis. However, the mechanism by which plastid retrograde signals and phytohormones cooperatively regulate plastid biogenesis remains elusive. Here, we investigated the effects of an inhibitor and a mutation that generate biogenic plastid signals on phytohormones and vice versa. Inhibition of plastid biogenesis by norflurazon (NF) treatment and the plastid protein import2 (ppi2) mutation caused a decrease in salicylic acid (SA) and jasmonic acid (JA). This effect can be attributed in part to the altered expression of genes involved in the biosynthesis and the metabolism of SA and JA. However, SA-dependent induction of the PATHOGENESIS-RELATED1 gene was virtually unaffected in NF-treated plants and the ppi2 mutant. Instead, the level of chlorophyll in these plants was partially restored by the exogenous application of SA. Consistent with this observation, the levels of some photosynthesis-associated proteins increased in the ppi2 and NF-treated plants in response to SA treatment. This regulation in true leaves seems to occur at the posttranscriptional level since SA treatment did not induce the expression of photosynthesis-associated genes. In salicylic acid induction deficient 2 and lesions simulating disease resistance 1 mutants, endogenous SA regulates the accumulation of photosynthesis-associated proteins through transcriptional and posttranscriptional mechanisms. These data indicate that SA acts antagonistically to the inhibition of plastid biogenesis by promoting the accumulation of photosynthesis-associated proteins in Arabidopsis, suggesting a possible link between SA and biogenic plastid signaling.

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.

Plant molecular farming, that is, using plants as hosts for production of therapeutic proteins and high-value compounds, has gained substantial interest in recent years. Chloroplasts in particular are an attractive subcellular compartment for expression of foreign genes. Here, we present a new method for transgene introduction and expression in chloroplasts that, unlike classically used approaches, does not require transgene insertion into the chloroplast genome. Instead, the transgene is amplified as a physically independent entity termed a 'minichromosome'. Amplification occurs in the presence of a helper protein that initiates the replication process via recognition of specific sequences flanking the transgene, resulting in accumulation of extremely high levels of transgene DNA. Importantly, we demonstrate that such amplified transgenes serve as a template for foreign protein expression, are maintained stably during plant development and are maternally transmitted to the progeny. These findings indicate that the minichromosome-based approach is an attractive tool for transgene expression in chloroplasts and for organelle genome engineering.

Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron-induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis Ferroportin 3 (FPN3) is an iron exporter dual-targeted to mitochondria and chloroplasts.FPN3is expressed in shoots regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots.fpn3mutants cannot grow as well as wild type under iron-deficient conditions and shoot iron levels are reduced infpn3mutants compared to wild type. ICP-MS measurements show that iron levels in the mitochondria and chloroplasts are increased relative to wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron deficientfpn3mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis.Significance statementIron homeostasis must be tightly controlled in the mitochondria and chloroplasts, but iron trafficking in these organelles is not fully understood. Our work suggests that FPN3 is an iron exporter required for maintaining proper iron levels in mitochondria and chloroplasts. Furthermore, FPN3 is necessary for the optimal growth and normal mitochondrial ultrastructure under iron deficiency. This study reveals the physiological role of FPN3 and advances our understanding of iron regulation in mitochondria and chloroplasts.

Carotenoids play vital roles in the coloration of plant tissues and organs, particularly fruits; however, the regulation of carotenoid metabolism in fruits during ripening is largely unknown. Here, we show that red light promotes fruit coloration by inducing accelerated degreening and carotenoid accumulation in kumquat fruits. Transcriptome profiling revealed that a NAC (NAM/ATAF/CUC2) family transcription factor, FcrNAC22, is specifically induced in red light-irradiated fruits. FcrNAC22 localizes to the nucleus, and its gene expression is up-regulated as fruits change color. Results from dual luciferase, yeast one-hybrid assays and electrophoretic mobility shift assays indicate that FcrNAC22 directly binds to, and activates the promoters of three genes encoding key enzymes in the carotenoid metabolic pathway. Moreover, FcrNAC22 overexpression in citrus and tomato fruits as well as in citrus callus enhances expression of most carotenoid biosynthetic genes, accelerates plastid conversion into chromoplasts, and promotes color change. Knock down of FcrNAC22 expression in transiently transformed citrus fruits attenuates fruit coloration induced by red light. Taken together, our results demonstrate that FcrNAC22 is an important transcription factor that mediates red light-induced fruit coloration via up-regulation of carotenoid metabolism.

In chloroplasts, thiol-dependent redox regulation is linked to light since the disulfide reductase activity of thioredoxins (Trxs) relies on photo-reduced ferredoxin (Fdx). Furthermore, chloroplasts harbor an NADPH-dependent Trx reductase (NTR) with a joint Trx domain, termed NTRC. The activity of these two redox systems is integrated by the redox balance of 2-Cys peroxiredoxin (Prx), which is controlled by NTRC. However, NTRC was proposed to participate in redox regulation of additional targets, prompting inquiry into whether the function of NTRC depends on its capacity to maintain the redox balance of 2-Cys Prxs or by direct redox interaction with chloroplast enzymes. To answer this, we studied the functional relationship of NTRC and 2-Cys Prxs by a comparative analysis of the triple Arabidopsis (Arabidopsis thaliana) mutant, ntrc-2cpab, which lacks NTRC and 2-Cys Prxs, and the double mutant 2cpab, which lacks 2-Cys Prxs. These mutants exhibit almost indistinguishable phenotypes: in growth rate, photosynthesis performance, and redox regulation of chloroplast enzymes in response to light and darkness. These results suggest that the most relevant function of NTRC is in controlling the redox balance of 2-Cys Prxs. A comparative transcriptomics analysis confirmed the phenotypic similarity of the two mutants and suggested that the NTRC-2-Cys Prxs system participates in cytosolic protein quality control. We propose that NTRC and 2-Cys Prxs constitute a redox relay, exclusive to photosynthetic organisms that fine-tunes the redox state of chloroplast enzymes in response to light and affects transduction pathways towards the cytosol

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.

Reactive oxygen species (ROS) generation within a cell is a natural process of certain sub-cellular components involved in redox reactions. Within a plant cell, chloroplasts are one of the major roofs for ROS generation. Plastid generated ROS molecules involve singlet oxygen (1 O2 ), superoxide radical (O2 - ), hydroxyl radical (OH• ) and hydrogen peroxide (H2 O2 ) which are produced mainly during photochemical reactions of photosynthesis and chlorophyll biosynthetic process. Under normal growth and developmental process, generated ROS molecules act as a secondary messenger controlling several metabolic reactions, however perturbed environmental conditions lead to multifold amplification of cellular ROS that eventually drags the target cell to death. To maintain the homeostasis between production and scavenging of ROS, the cell has instituted several enzymatic and non-enzymatic anti-oxidant machineries to bring ROS at the physiological level. Among chloroplastic ROS molecules, excessive generation of singlet oxygen (1 O2 ) is highly deleterious to the cells metabolic functions and in turn survival. Interestingly, within cellular antioxidant machinery, the enzymes involved in detoxification of 1 O2 are lacking. Recent studies suggest that under optimal concentration 1 O2 acts as a signaling molecule and drive the cell to either acclimation pathway or regulated cell death (RCD). Stress-induced RCD is one of the survival mechanisms for the whole plant and involvement of chloroplasts and chloroplasts localized molecules that execute RCD are not well understood. Through this review, we intend to advocate the participation of chloroplasts-generated 1 O2 in signaling and RCD in plants.

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.

Chlorophyll breakdown is a fundamental process in nature. The elucidation of the involved pathways was the outstanding scientific achievement of the late Stefan Hörtensteiner, to whom this paper is dedicated., With the finest biochemical and molecular approaches, convincing explorative strategies, and long-term vision, Stefan Hörtensteiner succeeded in elucidating the biochemical pathway responsible for chlorophyll degradation. After having contributed to the identification of key chlorophyll degradation products in the course of the past 25 years, he gradually identified and characterized most of the crucial players in the PAO/phyllobilin degradation pathway of chlorophyll. He was one of the brightest plant biochemists of his generation, and his work opened doors to a better understanding of plant senescence, tetrapyrrole homeostasis, and their complex regulation. He sadly passed away on 5 December 2020, aged 57.

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.

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).

Photosynthetic organisms have developed diverse antennas composed of chromophorylated proteins to increase photon capture. Cryptophyte algae acquired their photosynthetic organelles (plastids) from a red alga by secondary endosymbiosis. Cryptophytes lost the primary red algal antenna, the red algal phycobilisome, replacing it with a unique antenna composed of αβ protomers, where the β subunit originates from the red algal phycobilisome. The origin of the cryptophyte antenna, particularly the unique α subunit, is unknown. Here we show that the cryptophyte antenna evolved from a complex between a red algal scaffolding protein and phycoerythrin β. Published cryo-EM maps for two red algal phycobilisomes contain clusters of unmodelled density homologous to the cryptophyte-αβ protomer. We modelled these densities, identifying a new family of scaffolding proteins related to red algal phycobilisome linker proteins that possess multiple copies of a cryptophyte-α-like domain. These domains bind to, and stabilise, a conserved hydrophobic surface on phycoerythrin β, which is the same binding site for its primary partner in the red algal phycobilisome, phycoerythrin α. We propose that after endosymbiosis these scaffolding proteins outcompeted the primary binding partner of phycoerythrin β, resulting in the demise of the red algal phycobilisome and emergence of the cryptophyte antenna., Cryptophytes acquired plastids from red algae but replaced the light-harvesting phycobilisome with a unique cryptophyte antenna. Here via analysis of phycobilisome cryo-EM structures, Rathbone et al. propose that the α subunit of the cryptophyte antenna originated from phycobilisome linker proteins

Phytohormones are known to play an important role in the regulation of chloroplast biogenesis. However, information on the pathways of hormonal signal perception by chloroplasts is limited. In order to analyze the possible mechanisms of hormone action, the response to abscisic acid (ABA) and cytokinin (CKs) treatment of Arabidopsis thaliana (L.) Heynh. scabra3-2 (sca3-2) and rpotmp mutants with defective genes for nuclear-encoded plastid RNA polymerases was studied. The reaction of the rpotmp mutant usually did not differ from the response of wild-type plants: trans-zeatin activated the accumulation of transcripts of genes encoding the transcription apparatus and genes encoded by the plastid genome, while ABA weakly suppressed their expression or had no significant effect. However, the steady-state levels of gene templates of the plastid-encoded RNA polymerase (PEP-A) enzyme complex in the rpotmp mutant were higher than in wild-type plants, probably due to a compensatory mechanism that allows the mutant to enhance PEP-dependent transcription in the absence of the RPOTmp enzyme. Dysfunction of the plastid RNA polymerase RPOTp enzyme in the sca3-2 mutant induced an altered hormonal response of the genes for plastid transcriptional complex and plastid-encoded genes in comparison with the response of wild-type plants. The absence of a significant up-regulating effect of CKs on the expression of genes encoding the transcription apparatus correlated with the retention of the steady-state levels of transcripts for chloroplast genes and plastid proteins. In addition, the sca3-2 mutant had an increased steady-state expression of genes for CK metabolism: IPT3, IPT5, and CKX5. However, the response to the inhibitory effect of ABA in this mutant, on the contrary, was more pronounced, which is possibly caused by the changes in the steady-state levels of the transcripts of a number of genes encoding of ABA signaling and metabolism proteins. Thus, changes in the expression of NEP (nuclear-encoded RNA polymerases) genes under the action of hormones can be transformed into activation or inhibition in the expression of chloroplast genes of nuclear or plastome coding, providing the regulation of chloroplast functions through the mechanisms of anterograde signaling.

Transitory starch is the portion of starch that is synthesized during the day in the chloroplast and usually used for plant growth overnight. Here, we report altered metabolism of transitory starch in the wxr1/wxr3 (weak auxin response 1/3) mutants of Arabidopsis. WXR1/WXR3 were previously reported to regulate root growth of young seedlings and affect the auxin response mediated by auxin polar transport in Arabidopsis. In this study the wxr1/wxr3 mutants accumulated transitory starch in cotyledon, young leaf, and hypocotyl at the end of night. WXR1/WXR3 expression showed diurnal variation. Grafting experiments indicated that the WXRs in root were necessary for proper starch metabolism and plant growth. We also found that photosynthesis was inhibited and the transcription level of DIN1/DIN6 (Dark-Inducible 1/6) was reduced in wxr1/wxr3. The mutants also showed a defect in the ionic equilibrium of Na+ and K+, consistent with our bioinformatics data that genes related to ionic equilibrium were misregulated in wxr1. Loss of function of WXR1 also resulted in abnormal trafficking of membrane lipids and proteins. This study reveals that the plastid proteins WXR1/WXR3 play important roles in promoting transitory starch degradation for plant growth over night, possibly through regulating ionic equilibrium in the root.

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.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.

Based on morphological and molecular evidence, Luisia yunnanensis (Epidendroideae, Orchidaceae), a new species from Yunnan, China, is described and illustrated. A detailed comparison between the newly discovered orchid and other members of Luisia is presented. Molecular analysis of nrITS and plastid DNA (matK, rbcL) sequence data show that L. yunnanensis is sister to L. filiformis with strong support.

Barleria albostellata (Acanthaceae), is a valuable medicinal plant with a broad spectrum of antibacterial and anti-inflammatory activities. This study aimed to characterize the micromorphology, distribution, and chemical composition of the trichomes present on the leaves and stems of B. albostellata using light and electron microscopy and histochemistry. Morphological observations using stereo and scanning electron microscopy (SEM) revealed a dense indumentum bearing numerous non-glandular trichomes on the leaves and stems of B. albostellata. The histochemical and SEM analyses revealed the presence of five morphologically distinct glandular capitate trichome types, multangulate-dendritic branched (MDB) non-glandular trichomes and a glandular head attached to a branched non-glandular trichome. Transmission electron micrographs showed that numerous plastids, mitochondria, and endoplasmic reticulum cisternae were actively involved in the secretory process. The stained leaf and stem sections indicated the presence of alkaloids and phenolics as the major medicinal compounds in glandular and non-glandular trichomes. To our knowledge, this study represents the first detailed report describing the key micromorphological features of the foliar structures of B. albostellata as well as the preliminary chemical composition of the secretions produced by these structures.

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.

Reprogramming metabolism, in addition to modifying the structure and function of the photosynthetic machinery, is crucial for plant acclimation to changing light conditions. One of the key acclimatory responses involves reorganization of the photosynthetic membrane system including changes in thylakoid stacking. Glycerolipids are the main structural component of thylakoids and their synthesis involves two main pathways localized in the plastid and the endoplasmic reticulum (ER); however, the role of lipid metabolism in light acclimation remains poorly understood. We found that fatty acid synthesis, membrane lipid content, the plastid lipid biosynthetic pathway activity, and the degree of thylakoid stacking were significantly higher in plants grown under low light compared with plants grown under normal light. Plants grown under high light, on the other hand, showed a lower rate of fatty acid synthesis, a higher fatty acid flux through the ER pathway, higher triacylglycerol content, and thylakoid membrane unstacking. We additionally demonstrated that changes in rates of fatty acid synthesis under different growth light conditions are due to post-translational regulation of the plastidic acetyl-CoA carboxylase activity. Furthermore, Arabidopsis mutants defective in one of the two glycerolipid biosynthetic pathways displayed altered growth patterns and a severely reduced ability to remodel thylakoid architecture, particularly under high light. Overall, this study reveals how plants fine-tune fatty acid and glycerolipid biosynthesis to cellular metabolic needs in response to long-term changes in light conditions, highlighting the importance of lipid metabolism in light acclimation.

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.

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.

Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

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.

Crop plants, such as watermelon, suffer from severe Aluminum (Al3+)-toxicity in acidic soils with their primary root elongation being first arrested. However, the significance of apoplastic or symplastic Al3+-toxicity in watermelon root is scarcely reported. In this work, we identified a medium fruit type (ZJ) and a small fruit type (NBT) as Al+3-tolerant and sensitive based on their differential primary root elongation rate respectively, and used them to show the effects of symplastic besides apoplastic Al distribution in the watermelon's root. Although the Al content was higher in the root of NBT than ZJ, Al+3 allocated in their apoplast, vacuole and plastid fractions were not significantly different between the two cultivars. Thus, only a few proportion of Al+3 differentially distributed in the nucleus and mitochondria corresponded to interesting differential morphological and physiological disorders recorded in the root under Al+3-stress. The symplastic amount of Al+3 substantially induced the energy efficient catalase pathway in ZJ, and the energy consuming ascorbate peroxidase pathway in NBT. These findings coincided with obvious starch granule visibility in the root ultra-structure of ZJ than NBT, suggesting a differential energy was used in supporting the root elongation and nutrient uptake for Al+3-tolerance in the two cultivars. This work provides clues that could be further investigated in the identification of genetic components and molecular mechanisms associated with Al+3-tolerance in watermelon.

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.

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.

SUMMARY Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis-prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis-prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.

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.

In a mixotrophic Chlamydomonas reinhardtii culture, the expression levels of genes encoding primary metabolic enzymes and chloroplast plastid transporters were analyzed. For the majority of the genes studied, their expression levels decreased as they approached the final stages of culture growth. During the period of exponential growth, the expression profiles changed more intensively than during the stationary phase. In the middle of exponential growth, significant changes of mRNA profiles reflected reorganization of metabolism, with an emphasis on the induction of lipid synthesis, accompanied by alterations in carbon fluxes through biochemical pathways and a shift in the energy balance between the plastid and cytosol.

Plant organelle (plastid and mitochondrial) genomes contain substantial information for plant evolution and adaptation. Therefore, it’s important to reveal plant whole-genome sequences including plastid and mitochondrial genomes. To decode these sequences, it is required to efficiently separate organelle genomic DNA from nucleus genome, which is difficult and laborious. In this study, an efficient procedure was established to obtain plant organelle genomes without extraction of plastid and mitochondria. Organelle DNA was extracted from three materials including Sinapis arvensis var. ‘Yeyou 18’, a cytoplasmic male sterile line (Nsa CMS) and its corresponding maintainer line ‘Zhongshuang 4’. DNA was sequenced by Roche 454 FLX+ and Illumina Miseq platforms. Organelle genomes were assembled using the generated reads and public organelle genome sequences. This research presented a procedure that efficiently assembled organelle genomes and subsequent fill gaps by extending the consensus contig terminals. This method enabled us to assemble plant plastid and mitochondrial genomes simultaneously. The obtained organelle genomes could accelerate understanding of mitochondrial rearrangements and laid a foundation for further study of Sinapis arvensis evolution and sterility gene of Nsa CMS.

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.