Formation of Sb2O3 microcrystals by Rhodotorula mucilaginosa.

Antimony (Sb) pollution seriously endangers ecological environment and human health. Microbial induced mineralization can effectively convert metal ions into more stable and less soluble crystalline minerals by extracellular polymeric substance (EPS). In this study, an efficient Sb-resistant Rhodotorula mucilaginosa (R. mucilaginosa) was screened, which can resist 41 mM Sb(III) and directly transform Sb(III) into Sb2O3 microcrystals by EPS. The removal efficiency of R. mucilaginosa for 22 mM Sb(III) reached 70% by converting Sb(III) to Sb2O3. The components of supernatants as well as the effects of supernatants and pH on Sb(III) mineralization verified that inducible and non-inducible extracellular protein/polysaccharide biomacromolecules play important roles in the morphologies and sizes control of Sb2O3 formed by R. mucilaginosa respectively. Sb2O3 microcrystals with different morphologies and sizes can be prepared by the regulation of inducible and non-inducible extracellular biomacromolecules secreted by R. mucilaginosa. This is the first time to identify that R. mucilaginosa can remove Sb(III) by transforming Sb(III) into Sb2O3 microcrystals under the control of EPS. This study contributes to our understanding for Sb(III) biomineralization mechanisms and provides strategies for the remediation of Sb-contaminated environment.

The highly effective cadmium-resistant mechanism of Pseudomonas aeruginosa and the function of pyoverdine induced by cadmium.

Pyoverdine (PVD) plays an important role in reducing cadmium (Cd) accumulation in plants. Some Pseudomonas aeruginosa (P. aeruginosa) species can produce PVD under Cd(Π) stress. However, the function of Cd(Π)-induced PVD remains unclear. In this study, we isolated a highly effective Cd(Π)-resistant P. aeruginosa which can secrete PVD under Cd(Π) stress and found that PVD secretion has a dose-dependent relationship with Cd(Π) concentration. PVD can form a PVD-Cd complex with Cd(Π), though the PVD-Cd complex is unable to be adsorbed by the cell or enter the cell, so the complexation of PVD and Cd(Π) impedes Cd(Π) adsorption on the cell surface and alleviates the oxidative stress, lipid peroxidation, and morphological destruction of the cell caused by Cd(Π) and effectively improves the resistance of P. aeruginosa to Cd(Π). In summary, our research results indicate that the Cd(Π) resistance mechanism of P. aeruginosa screened is the complexation of PVD for Cd(Π) and the adsorption of bacteria for Cd(Π); furthermore, PVD plays an important role in improving the Cd(Π)-resistant ability of bacteria. This study provides a deeper understanding of the highly effective Cd(Π) resistance mechanism of P. aeruginosa and the function of Cd(Π)-induced PVD in bacteria.

Graphene oxide quantum dots (GOQDs) induce behavioral disorders via the disturbance of kynurenine pathway in zebrafish larvae.

The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.

mitfa deficiency promotes immune vigor and potentiates antitumor effects in zebrafish.

The mitfa gene is a well-known transcription factor associated with microphthalmia and is essential for early melanophore development. However, little is known about how mitfa affects the immune system. Here, we generated a novel mitfa knock-out zebrafish line using the CRISPR/Cas9 system. The mitfa-/- zebrafish exhibited reduced melanin levels compared to the nacre mutant. We investigated the impact on the immune system after exposure to Edwardsiella tarda and bifenazate in zebrafish larvae, and observed that the macrophage numbers were reduced in both treated groups. Remarkably, the expression levels of immune-related genes exhibited significant increases after bacterial challenge or bifenazate exposure in the mitfa-/- zebrafish, except for tlr4 and rela. Furthermore, we conducted xenograft experiments using mouse B16 melanoma cells. Notably, the cancer cells didn't show a high cell migration ratio, implying that the immune system was highly activated after the loss of mifta. Taken together, our findings suggest that mitfa-/- zebrafish serve as a valuable model for investigating the relationship between the immune system and melanocytes, providing new insights into the role of mitfa in immune responses.

Graphene quantum dots (GQDs) induce thigmotactic effect in zebrafish larvae via modulating key genes and metabolites related to synaptic plasticity.

Graphene quantum dots (GQDs) recently gain much attention for its medicinal values in treating diseases such as neurodegeneration and inflammations. However, owing to the high permeability of GQDs across the blood-brain barrier, whether its retention in the central nervous system (CNS) perturbs neurobehaviors remains less reported. In the study, the locomotion of zebrafish larvae (Danio rerio) was fully evaluated when administrated by two GQDs in a concentration gradient, respectively as reduced-GQDs (R-GQDs): 150, 300, 600, 1200, and 2400 g/L, and graphene oxide QDs (GOQDs): 60, 120, 240, 480, and 960 g/L. After exposure, the larvae were kept for locomotion analysis within one week's depuration. Substantial data showed that the basal locomotor activity of zebrafish larvae was not significantly changed by both two GQDs at low concentrations while weakened greatly with the increase of concentrations, and the total ATP levels of zebrafish larvae were also found to decrease significantly when exposed to the highest concentrations of GQDs. Next, the thigmotactic effect was observed to be remarkably induced in larvae by both two GQDs at any concentrations during exposure, and remained strong in larvae treated by high concentrations of R-GQDs after 7 days' depuration. To be noted, we found that GQDs affected the synaptic plasticity via downregulating the mRNA levels of NMDA and AMPA receptor family members as well as the total glutamine levels in zebrafish larvae. Together, our study presented robust data underlying the locomotor abnormalities aroused by GQDs in zebrafish larvae and indicated the potential adverse effects of GQDs on synaptic plasticity.

Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish.

MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17-/- zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17-/- larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17-/- zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17-/- zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17-/- zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17-/- zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.

Transcriptome and in silico approaches provide new insights into the mechanism of male reproductive toxicity induced by chronic exposure to DEHP.

Di-(2-ethylhexyl) phthalate (DEHP) can affect the male reproductive system in vertebrates, but the underlying molecular mechanism is still elusive. Therefore, in this study, we aimed to dig the in-depth mechanism of DEHP-induced reproductive toxicity on male zebrafish via testicular transcriptome using embryo exposed at the environmentally relevant concentration (ERC) of 100 µg/L for 111 days. Moreover, our results were further confirmed via in silico technique and bioassay experimental in vitro (cell lines) and in vivo (zebrafish). The results showed DEHP exposure could affect male spermatogenesis, altered gonad histology, and reduced egg fertilization rate. Transcriptome analysis identified 1879 significant differentially expressed genes enriched in the exposure group. Twenty-seven genes related to three pathways of reproduction behavior were further validated by qPCR. In silico molecular docking revealed that DEHP and its metabolism bind to the zebrafish progesterone receptor (Pgr), suggesting the potential disruption of DEHP to the normal Pgr signaling. To further validate it, a wild-type Pgr plasmid and its mutants on specific binding sites were constructed. The transfection and microinjection experiment demonstrated that these binding sites mutations of Pgr affected the expression levels of male reproductive toxicity. Taken together, our study provided new insight into the molecular mechanisms of male reproductive toxicity induced by DEHP, and Pgr may serve as an important target binding by DEHP pollution, which needs further study in the future.

Generation of a novel transgenic marine medaka (Oryzias melastigma) for highly sensitive detection of heavy metals in the environment.

As typic priority pollutants in the marine environment, heavy metals can be accumulated in the human body leading to serious environmental and health problems. The metal regulatory elements (MREs) have been identified to be the main functional parts for the response to heavy metals. To develop a convenient biological monitoring tool for the detection of heavy metals in the oceans, we generated a transgenic marine medaka line Tg(OmMT: eGFP) with a truncated metallothionein promoter, which was only 193 bp and drove the expression of eGFP. After Tg(OmMT:eGFP) embryos were treated with four different heavy metals and different concentrations, the results showed that the expression level of eGFP was consistent with that of the endogenous mt. The transgenic embryos are very sensitive to Hg2+, and the fluorescence could be induced in the 0.0002 µM concentration, which is far lower than the primary water standard. The expression level of eGFP and mt showed a dose-dependent manner to heavy metals concentration. Taken together, the newly established marine medaka is a sensitive, efficient, and convenient tool for monitoring heavy metal pollution in the environment, especially seawater.

Role of manganese superoxide dismutase (Mn-SOD) against Cr(III)-induced toxicity in bacteria.

The toxicity of Cr(VI) was widely investigated, but the defense mechanism against Cr(III) in bacteria are seldom reported. Here, we found that Cr(III) inhibited bacterial growth and induced reactive oxygen species (ROS). After exposure to Cr(III), loss of sodA not only led to the excessive generation of ROS, but also enhanced the level of lipid peroxidation and reduced the GSH level, indicating that the deficiency of Mn-SOD decreased the bacterial resistance ability against Cr(III). The adverse effects of oxidative stress caused by Cr(III) could be recovered by the rescue of Mn-SOD in the sodA-deficient strain. Besides the oxidative stress, Cr(III) could cause the bacterial morphology variation, which was distinct between the wild-type and the sodA-deficient strains due to the differential expressions of Z-ring division genes. Moreover, Mn-SOD might prevent Cr(III) from oxidation on the bacterial surface by combining with Cr(III). Taken together, our results indicated that the Mn-SOD played a vital role in regulating the stress resistance, expression of cell division-related genes, bacterial morphology, and chemistry valence state of Cr. Our findings firstly provided a more in-depth understanding of Cr(III) toxicity and bacterial defense mechanism against Cr(III).

Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish.

The molecular pathogenesis of glioblastoma indicates that RTK/Ras/PI3K, RB and TP53 pathways are critical for human gliomagenesis. Here, several transgenic zebrafish lines with single or multiple deletions of nf1, tp53 and rb1 in astrocytes, were established to genetically induce gliomagenesis in zebrafish. In the mutant with a single deletion, we found only the nf1 mutation low-efficiently induced tumour incidence, suggesting that the Nf1 pathway is critical for the initiation of gliomagenesis in zebrafish. Combination of mutations, nf1;tp53 and rb1;tp53 combined knockout fish, showed much higher tumour incidences, high-grade histology, increased invasiveness, and shortened survival time. Further bioinformatics analyses demonstrated the alterations in RTK/Ras/PI3K, cell cycle, and focal adhesion pathways, induced by abrogated nf1, tp53, or rb1, were probably the critical stepwise biological events for the initiation and development of gliomagenesis in zebrafish. Gene expression profiling and histological analyses showed the tumours derived from zebrafish have significant similarities to the subgroups of human gliomas. Furthermore, temozolomide treatment effectively suppressed gliomagenesis in these glioma zebrafish models, and the histological responses in temozolomide-treated zebrafish were similar to those observed in clinically treated glioma patients. Thus, our findings will offer a potential tool for genetically investigating gliomagenesis and screening potential targeted anti-tumour compounds for glioma treatment.

Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System.

The mitochondria DNA (mtDNA) editing tool, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system, is a promising approach for the treatment of mtDNA diseases by eliminating mutant mitochondrial genomes. However, there have been no reports of repairing the mutant mtDNA with homologous recombination strategy to date. Here, we show a mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and reduce mtDNA copy number in both human cells and zebrafish. An exogenous single-stranded DNA with short homologous arm was knocked into the targeting loci accurately, and this mutagenesis could be steadily transmitted to F1 generation of zebrafish. Moreover, we found some major factors involved in nuclear DNA repair were upregulated significantly by the mito-CRISPR/Cas9 system. Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a useful method to edit mtDNA by knock-in strategy, providing a potential therapy for the treatment of inherited mitochondrial diseases.

Nitrogen-doped graphene quantum dots (N-GQDs) perturb redox-sensitive system via the selective inhibition of antioxidant enzyme activities in zebrafish.

Graphene quantum dots (GQDs) are well-known for its potential applications for bioimaging, biosensor, and drug carrier in biomedicine. GQDs are well characteristic of intrinsic peroxidase-like catalytic activity, which is proven effective in scavenging the free radicals, such assuperoxide anion, hydrogen peroxide, and hydroxyl radical. GQDs are also well praised for its low in vivo and in vitro toxicity. Here, we found that nitrogen-doped GQDs (N-GQDs) can strongly disturb redox-sensitive system via the selective inhibition of endogenous antioxidant enzyme activities in zebrafish. The enzyme activities or transcription levels of a battery of hemoproteins including catalase (CAT), superoxide dismutase (SOD), respiratory chain complex I, complex Ⅲ, hemoglobin (Hb), and myeloperoxidase (MPO), were significantly suppressed by N-GQDs. We also found that N-GQDs activated the cytochrome P450 monooxygenase (e.g. cyp1a) and the associated aryl-hydrocarbon receptor repressors (ahrr1 and ahrr2) in zebrafish embryos. Compared to the ultrasmall graphene oxide (USGO), N-GQDs exhibited stronger fluorescent permeability and tissue-specific bio-accumulative effects. Taken together, our findings highlighted that exposure to N-GQDs can disrupt endogenous antioxidant enzyme activities, possibly via the competitive inhibition of electron transfer process. Our results in this study provided solid data for biosafety evaluations of various types of GQDs, and created an alert for the future biomedical applications of N-GQDs.

New toxicogenetic insights and ranking of the selected pharmaceuticals belong to the three different classes: A toxicity estimation to confirmation approach.

Tetracycline hydrochloride (TH), indomethacin (IM), and bezafibrate (BF) belong to the three different important classes of pharmaceuticals, which are well known for their toxicity and environmental concerns. However, studies are still elusive to highlight the mechanistic toxicity of these pharmaceuticals and rank them using both, the toxicity prediction and confirmation approaches. Therefore, we employed the next generation toxicity testing in 21st century (TOX21) tools and estimated the in vitro/vivo toxic endpoints of mentioned pharmaceuticals, and then confirmed them using in vitro/vivo assays. We found significant resemblance in the results obtained via both approaches, especially in terms of in vivo LC50 s and developmental toxicity that ranked IM as most toxic among the studied pharmaceuticals. However, TH appeared most toxic with the lowest estimated AC50s, the highest experimental IC50s, and DNA damages in vitro. Contrarily, IM was found as congener with priority concern to activate the Pi3k-Akt-mTOR pathway in vitro at concentrations substantially lower than that of TH and BF. Further, IM exposure at lower doses (2.79-13.97 µM) depressed the pharmaceuticals detoxification phase I (CYP450 s), phase II (UGTs, SULTs), and phase III (TPs) pathways in zebrafish, whereas, at relatively higher doses, TH (2.08-33.27 µM) and BF (55.28-884.41 µM) partially activated these pathways, which ultimately caused the developmental toxicity in the following order: IM > TH > BF. In addition, we also ranked these pharmaceuticals in terms of their particular toxicity to myogenesis, hematopoiesis, and hepatogenesis in zebrafish embryos. Our results revealed that IM significantly affected myogenesis, hematopoiesis, and hepatogenesis, while TH and BF induced prominent effects on hematopoiesis via significant downregulation of associated genetic markers, such as drl, mpx, and gata2a. Overall, our findings confirmed that IM has higher toxicity than that of TH and BF, therefore, the consumption of these pharmaceuticals should be regulated in the same manner to ensure human and environmental safety.

CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.

Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.

Deletion of mstna and mstnb impairs the immune system and affects growth performance in zebrafish.

Myostatin (Mstn) is a negative regulator of muscle development in vertebrates. Although its function in muscle growth has been well studied in mammals and fish, it remains unclear whether or how mstn functions in the immune system. In this study, mstna-/- and mstnb-/- homozygous zebrafish were firstly generated using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). Deletion of mstnb but not mstna enhanced growth performance. Although survival rates under normal conditions were slightly decreased in both strains, mortality after dexamethasone-induced stress was increased by ∼30%. Furthermore, transcriptional levels of several critical immune-related genes were decreased, and the ability to withstand exposure to pathogenic E. tarda was decreased, compared with that of controls. In mstnb-/- but not mstna-/- zebrafish, expression of NF-κB subunits and several pro-inflammatory cytokines failed to respond to E. tarda exposure except nfkb1, c-rel and tnfα. Taken together, these results indicate that mstnb but not mstna plays a key role in zebrafish muscle growth. While each paralogue contributes to the response to bacterial insult, mstnb affects the immune system through activation of the NF-κB pathway, and mstna is likely to act upstream of NF-κB at some as yet unidentified target.

Generation and application of a novel transgenic zebrafish line Tg(cyp1a:mCherry) as an in vivo assay to sensitively monitor PAHs and TCDD in the environment.

The polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) are classified as human carcinogens, and can also cause serious health problems. To develop a convenient bio-monitoring tool for the detection of PAHs and TCDD in the environment, we generated a transgenic zebrafish line Tg(cyp1a:mCherry) with cyp1a promoter driving mCherry expression. Here, Tg(cyp1a:mCherry) embryos were treated with different concentrations of TCDD and five US EPA priority PAHs congeners. The results showed that the expressions of mCherry and endogenous cyp1a were consistent with the PAHs exposure concentrations and were largely induced by TCDD and ≥4-ring PAHs. Moreover, the sensitivity of Tg(cyp1a:mCherry) embryos was also evaluated through monitoring of the PAHs contamination in the water and soil samples. The elevated red fluorescent signals and cyp1a expression levels were observed in Tg(cyp1a:mCherry) zebrafish after exposure to water samples and soil organic extracts with higher concentrations of ≥4-ring PAHs. These results further strengthen our findings of concentration- and congener-dependent response of the newly established zebrafish. Taken together, the newly established zebrafish line will prove as a sensitive, efficient and convenient tool for monitoring PAHs and TCDD contamination in the environment.

A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system.

Zebrafish as a model system to study toxicology.

Monitoring and assessing the effects of contaminants in the aquatic eco-environment is critical in protecting human health and the environment. The zebrafish has been widely used as a prominent model organism in different fields because of its small size, low cost, diverse adaptability, short breeding cycle, high fecundity, and transparent embryos. Recent studies have demonstrated that zebrafish sensitivity can aid in monitoring environmental contaminants, especially with the application of transgenic technology in this area. The present review provides a brief overview of recent studies on wild-type and transgenic zebrafish as a model system to monitor toxic heavy metals, endocrine disruptors, and organic pollutants for toxicology. The authors address the new direction of developing high-throughput detection of genetically modified transparent zebrafish to open a new window for monitoring environmental pollutants.

Proteomic analysis of chloroplast-to-chromoplast transition in tomato reveals metabolic shifts coupled with disrupted thylakoid biogenesis machinery and elevated energy-production components.

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.