Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages.

Industrial microalgae are promising photosynthetic cell factories, yet tools for large-scale targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica, we established a method to precisely and serially delete large genome fragments of ~100 kb from its 30.01 Mb nuclear genome. We started by identifying the 'non-essential' chromosomal regions (i.e. low expression region or LER) based on minimal gene expression under N-replete and N-depleted conditions. The largest such LER (LER1) is ~98 kb in size, located near the telomere of the 502.09-kb-long Chromosome 30 (Chr 30). We deleted 81 kb and further distal and proximal deletions of up to 110 kb (21.9% of Chr 30) in LER1 by dual targeting the boundaries with the episome-based CRISPR/Cas9 system. The telomere-deletion mutants showed normal telomeres consisting of CCCTAA repeats, revealing telomere regeneration capability after losing the distal part of Chr 30. Interestingly, the deletions caused no significant alteration in growth, lipid production or photosynthesis (transcript-abundance change for < 3% genes under N depletion). We also achieved double-deletion of both LER1 and LER2 (from Chr 9) that total ~214 kb at maximum, which can result in slightly higher growth rate and biomass productivity than the wild-type. Therefore, loss of the large, yet 'non-essential' regions does not necessarily sacrifice important traits. Such serial targeted deletions of large genomic regions had not been previously reported in microalgae, and will accelerate crafting minimal genomes as chassis for photosynthetic production.

Single-Cell Identification, Drug Susceptibility Test, and Whole-genome Sequencing of Helicobacter pylori Directly from Gastric Biopsy by Clinical Antimicrobial Susceptibility Test Ramanometry.

BACKGROUND: The battle against Helicobacter pylori (H. pylori) infections demands fast, reliable, and sensitive methods for pathogen identification (ID), antimicrobial susceptibility tests (ASTs) based on metabolic response, and genome-wide mutation profiling that reveals resistance mechanisms. METHODS: Here we introduce Clinical Antimicrobial Susceptibility Test Ramanometry for H. pylori (CAST-R-HP), and its validation with clinical samples. This method performs rapid ID, metabolism inhibition-based AST, and high-quality whole-genome sequencing for cells of targeted resistance phenotype, all at precisely 1-cell resolution and directly from biopsy samples. RESULTS: In CAST-R-HP, automated acquisition and machine learning of single-cell Raman spectra (SCRS) enable distinguishing individual H. pylori cells directly from a biopsy sample, with 98.5 ± 0.27% accuracy in ID. Moreover, by adding a 48- to72-h D2O feeding and drug exposure step prior to SCRS acquisition, CAST-R-HP reports AST for levofloxacin and clarithromycin with 100% accuracy, based on metabolic inhibition level. Furthermore, CAST-R-HP supports rapid sorting, low-bias DNA amplification, and full genome sequencing of single H. pylori cells with the SCRS defined, targeted drug-susceptibility phenotype, via Raman-activated gravity-driven cell encapsulation and sequencing. The genome-wide mutation map (maximum 99.70% coverage), at precisely 1-cell resolution, not only elucidates the drug-susceptibility phenotypes but also unveils their underlying molecular mechanisms. CONCLUSION: The culture independency, shorter turnaround time, high resolution, and comprehensive information output suggest that CAST-R-HP is a powerful tool for diagnosing and treating H. pylori infections.

The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

Integrated Addressable Dynamic Droplet Array (aDDA) as Sub-Nanoliter Reactors for High-Coverage Genome Sequencing of Single Yeast Cells.

An addressable dynamic droplet array (aDDA) is presented that combines the advantages of static droplet arrays and continuous-flow droplet platforms. Modular fabrication is employed to create a self-contained integrated aDDA. All the sample preparation steps, including single-cell isolation, cell lysis, amplification, and product retrieval, are performed in sequence within a sub-nanoliter (≈300 pL) droplet. Sequencing-based validation suggests that aDDA reduces the amplification bias of multiple displacement amplification (MDA) and elevates the percentage of one-yeast-cell genome recovery to 91%, as compared to the average of 26% using conventional, 20 µL volume MDA reactions. Thus, aDDA is a valuable addition to the toolbox for high-genome-coverage sequencing of single microbial cells.

Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.

In Raman-activated cell ejection and sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE Escherichia coli cells from <20% to ∼50%, while greatly improving the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance and pinpoint protection of cells from laser irradiation as a priority in method development.

Phenome-Genome Profiling of Single Bacterial Cell by Raman-Activated Gravity-Driven Encapsulation and Sequencing.

The small size and low DNA amount of bacterial cells have hindered establishing phenome-genome links in a precisely indexed, one-cell-per-reaction manner. Here, Raman-Activated Gravity-driven single-cell Encapsulation and Sequencing (RAGE-Seq) is presented, where individual cells are phenotypically screened via single-cell Raman spectra (SCRS) in an aquatic, vitality-preserving environment, then the cell with targeted SCRS is precisely packaged in a picoliter microdroplet and readily exported in a precisely indexed, "one-cell-one-tube" manner. Such integration of microdroplet encapsulation to Raman-activated sorting ensures high-coverage one-cell genome sequencing or cultivation that is directly linked to metabolic phenotype. For clinical Escherichia coli isolates, genome assemblies derived from precisely one cell via RAGE-Seq consistently reach >95% coverage. Moreover, directly from a urine sample of urogenital tract infection, metabolic-activity-based antimicrobial susceptibility phenotypes and genome sequence of 99.5% coverage are obtained simultaneously from precisely one cell. This single-cell global mutation map corroborates resistance phenotype and genotype, and unveils epidemiological features with high specificity and sensitivity. The ability to profile and correlate bacterial metabolic phenome and high-quality genome sequences at one-cell resolution suggests broad application of RAGE-Seq.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis.

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3' untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.

Corrigendum: Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

[This corrects the article DOI: 10.3389/fbioe.2023.1233856.].

Single-cell rapid identification, in situ viability and vitality profiling, and genome-based source-tracking for probiotics products.

Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low-cost strategies for quality assessment. Here, we introduce a culture-free, one-cell-resolution, phenome-genome-combined strategy called Single-Cell Identification, Viability and Vitality tests, and Source-tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D2O-probed single-cell Raman spectrum (SCRS) enables species-level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C-D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source-tracking, single-cell Raman-activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one-cell-based genome assemblies that can reach ~99.40% genome-wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20-fold faster, >10-time cheaper, vitality-revealing, heterogeneity-resolving, and automation-prone, SCIVVS is a new technological and data framework for quality assessment of live-cell products.

[Correlation between psoas muscle area and contralateral hip fracture after intertrochanteric fracture].

OBJECTIVE: To investigate the relationship between the area of psoas major muscle(PMI) and recurrent contralateral hip fracture in the initial intertrochanteric fracture. METHODS: Total of 87 patients with intertrochanteric fracture of femur from January 2008 to January 2011 were selected for CT scanning of lumbar spine and hip at the time of the first fracture, and then divided into two groups according to whether there was fracture in the contralateral hip, 13 patients in the contralateral hip fracture group, 5 males and 8 females, aged(82.30±5.66) years;there were 74 cases in the non contralateral hip fracture group, including 32 males and 42 females, with an age of (79.70±5.84) years. The gender, age, preoperative blood albumin value, operation side, body mass index(BMI), Harris score of hip joint one year after operation, Barthel index before operation and medical diseases before operation were observed and compared between two groups. The PMI was used to compare the area of psosa major on CT before operation in two groups, and the correlation between the area of PMI at the time of initial fracture and the fracture of the contralateral hip was evaluated. RESULTS: The two groups were followed up for more than 2 years after operation. There was a significant difference in PMI between two groups(P<0.05). There was a significant positive correlation between preoperative PMI and the time of re fracture of the contralateral hip(r=0.641, P=0.018). CONCLUSION: There are differences in the area of PMI in patients with contralateral hip fracture, so the area of PMI can be regarded as an important risk factor for contralateral hip fracture.

[Comparison of curative effect between F-shaped hollow screw and traditional three parallel screws in the treatment of Pauwels type â ¢ femoral neck fracture].

OBJECTIVE: To analyze and compare the clinical efficacy of F-shaped hollow screw and traditional inverted triangle three parallel screws in the treatment of young and middle-aged Pauwels type Ⅲ femoral neck fracture. METHODS: From January 2017 to January 2020, 38 patients with Pauwels type Ⅲ femoral neck fracture were treated. They were divided into two groups according to different screw placement methods. Among them, 18 patients in group A were fixed with F-shaped hollow screw, including 12 males and 6 females, aged 37 to 55 years, the time from injury to operation was 1 to 3 days. Other 20 cases in group B were fixed with 3 parallel screws in traditional inverted triangle, including 12 males and 8 females, aged 35 to 55 years. The time from injury to operation was 1 to 3 days. The fracture nonunion, femoral head necrosis, femoral neck shortening, hollow screw withdrawal, hip function Harris score and visual analogue scale(VAS) of pain were compared between the two groups. RESULTS: All patients were followed up for 15 to 31 months. There was no significant difference in fracture nonunion, femoral neck shortening and femoral head necrosis between two groups(P>0.05). There was significant difference in screw withdrawal between two groups(P<0.05). There was no significant difference in hip Harris score and VAS between the two groups at 12 months after operation(P>0.05). CONCLUSION: The short-term and medium-term effects of F-shaped and traditional inverted triangle three parallel screws in the treatment of young and middle-aged Pauwels Ⅲ femoral neck fractures are similar, but the nail withdrawal rate of F-shaped hollow screw is low.

Revealing CO2-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics.

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO2-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO2-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their 13C-bicarbonate (13C-HCO3-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates 13C-HCO3-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO2 fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling.

Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature.

Due to the challenges in detecting in situ activity and cultivating the not-yet-cultured, functional assessment and mining of living microbes from nature has typically followed a 'culture-first' paradigm. Here, employing phosphate-solubilizing microbes (PSM) as model, we introduce a 'screen-first' strategy that is underpinned by a precisely one-cell-resolution, complete workflow of single-cell Raman-activated Sorting and Cultivation (scRACS-Culture). Directly from domestic sewage, individual cells were screened for in-situ organic-phosphate-solubilizing activity via D2O intake rate, sorted by the function via Raman-activated Gravity-driven Encapsulation (RAGE), and then cultivated from precisely one cell. By scRACS-Culture, pure cultures of strong organic PSM including Comamonas spp., Acinetobacter spp., Enterobacter spp. and Citrobacter spp., were derived, whose phosphate-solubilizing activities in situ are 90-200% higher than in pure culture, underscoring the importance of 'screen-first' strategy. Moreover, employing scRACS-Seq for post-RACS cells that remain uncultured, we discovered a previously unknown, low-abundance, strong organic-PSM of Cutibacterium spp. that employs secretary metallophosphoesterase (MPP), cell-wall-anchored 5'-nucleotidase (encoded by ushA) and periplasmic-membrane located PstSCAB-PhoU transporter system for efficient solubilization and scavenging of extracellular phosphate in sewage. Therefore, scRACS-Culture and scRACS-Seq provide an in situ function-based, 'screen-first' approach for assessing and mining microbes directly from the environment.

[Analysis of risk factors of perioperative blood transfusion in the treatment of femoral intertrochanteric fracture with proximal femoral nail antirotation].

OBJECTIVE: To explore the influencing factors of perioperative blood transfusion in the treatment of elderly femoral intertrochanteric fractures with proximal femoral nail antirotation(PFNA). METHODS: The clinical data of 109 elderly patients with intertrochanteric fractures who received PFNA treatment from July 2018 to January 2020 were retrospectively analyzed. Both pelvic hip X-rays and CT plain scans were performed before surgery. All patients were diagnosed by X-ray and CT plain scan of pelvis and hip before operation. Through the statistical analysis of the basic data of patients before and during operation, the risk factors of perioperative blood transfusion were explored. RESULTS: Logistic regression analysis showed that age (P=0.013), fracture type (P<0.01), diabetes history (P=0.031) and preoperative hemoglobin (P<0.01) were independent risk factors for perioperative blood transfusion in the treatment of intertrochanteric fractures in elderly patients with 109 patients. Spearman correlation analysis showed that there was a positive correlation between blood transfusion and age of patients (P= 0.017), fracture type (P<0.01), diabetes history (P=0.023), and negatively correlated with preoperative hemoglobin (P<0.01). However, gender (P=0.297), history of hypertension (P=0.318) and operation time(P=0.325) had no significant relationship with perioperative blood transfusion. CONCLUSION: Age, fracture type, diabetes history, and preoperative hemoglobin are independent risk factors for perioperative blood transfusion in the treatment of elderly intertrochanteric fractures with PFNA. The older the patient, the history of diabetes, the more unstable the fracture, and the lower preoperative hemoglobin, the more likely it is to require a blood transfusion, which may provide a reference for clinical perioperative blood transfusion decisions.

One-Cell Metabolic Phenotyping and Sequencing of Soil Microbiome by Raman-Activated Gravity-Driven Encapsulation (RAGE).

Soil harbors arguably the most metabolically and genetically heterogeneous microbiomes on Earth, yet establishing the link between metabolic functions and genome at the precisely one-cell level has been difficult. Here, for mock microbial communities and then for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) platform, which identifies, sorts, and sequences precisely one bacterial cell via its anabolic (incorporating D from heavy water) and physiological (carotenoid-containing) functions. We showed that (i) metabolically active cells from numerically rare soil taxa, such as Corynebacterium spp., Clostridium spp., Moraxella spp., Pantoea spp., and Pseudomonas spp., can be readily identified and sorted based on D2O uptake, and their one-cell genome coverage can reach ∼93% to allow high-quality genome-wide metabolic reconstruction; (ii) similarly, carotenoid-containing cells such as Pantoea spp., Legionella spp., Massilia spp., Pseudomonas spp., and Pedobacter spp. were identified and one-cell genomes were generated for tracing the carotenoid-synthetic pathways; and (iii) carotenoid-producing cells can be either metabolically active or inert, suggesting culture-based approaches can miss many such cells. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at exactly one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems. IMPORTANCE Soil is home to an enormous and complex microbiome that features arguably the highest genomic diversity and metabolic heterogeneity of cells on Earth. Their in situ metabolic activities drive many natural processes of pivotal ecological significance or underlie industrial production of numerous valuable bioactivities. However, pinpointing "who is doing what" in a soil microbiome, which consists of mainly yet-to-be-cultured species, has remained a major challenge. Here, for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) method, which identifies, sorts, and sequences at the resolution of precisely one microbial cell via its catabolic and anabolic functions. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems.

Integration of proteome and transcriptome refines key molecular processes underlying oil production in Nannochloropsis oceanica.

BACKGROUND: Under nitrogen deficiency situation, Nannochloropsis spp. accumulate large amounts of lipids in the form of triacylglycerides (TAG). Mechanisms of this process from the perspective of transcriptome and metabolome have been obtained previously, yet proteome analysis is still sparse which hinders the analysis of dynamic adaption to nitrogen deficiency. Here, proteomes for 3 h, 6 h, 12 h, 24 h, 48 h and 10th day of nitrogen deplete (N-) and replete (N+) conditions were obtained and integrated with previous transcriptome data for N. oceanica. RESULTS: Physiological adaptations to N- not apparent from transcriptome data were unveiled: (a) abundance of proteins related to photosynthesis only slightly decreased in the first 48 h, indicating that photosynthesis is still working efficiently, and protein amounts adjust gradually with reduction in chloroplast size. (b) Most proteins related to the TCA cycle were strongly upregulated after 48 h under N-, suggesting that respiration is enhanced after 48 h and that TCA cycle efflux supports the carbon required for lipid synthesis. (c) Proteins related to lipid accumulation via the Kennedy pathway increased their abundance at 48 h, synchronous with the previously reported diversification of fatty acids after 48 h. CONCLUSIONS: This study adds a proteome perspective on the major pathways for TAG accumulation in Nannochloropsis spp. Temporal changes of proteome exhibited distinct adaptation phases that are usually delayed relative to transcriptomic responses. Notably, proteome data revealed that photosynthesis and carbon fixation are still ongoing even after 48 h of N-. Moreover, sometimes completely opposite trends in proteome and transcriptome demonstrate the relevance of underexplored post-transcriptional regulation for N- adaptation.

Transcriptomic and proteomic choreography in response to light quality variation reveals key adaption mechanisms in marine Nannochloropsis oceanica.

Photosynthetic organisms need to respond frequently to the fluctuation of light quality and light quantity in their habitat. In response to the fluctuation of different single wavelength lights, these organisms have to adjust and optimize the employment of light energy by redistributing excitation energy and remodeling photosystem stoichiometry or light complex structure. However, the response of whole cellular processes to fluctuations in single wavelength light is mostly unknown. Here, we report the transcriptomic and proteomic dynamics and metabolic adaptation mechanisms of Nannochloropsis oceanica to blue and red light. Preferential exposure to different light spectra induces massive reprogramming of the Nannochloropsis transcriptome and proteome. Combined with physiological and biochemical investigation, the rewiring of many cellular processes was observed, including carbon/nitrogen assimilation, photosynthesis, chlorophyll and cartenoid biosynthesis, reactive oxygen species (ROS) scavenging systems, and chromatin state regulation. A strong and rapid regulation of genes or proteins related to nitrogen metabolism, photosynthesis, chlorophyll synthesis, ROS scavenging system, and carotenoid metabolism were observed during 12 h and 24 h of exposure under red light. Additionally, two light harvesting complex proteins induced by blue light and one by red light were observed. The differential responses of N. oceanica to red and blue irradiation reveal how marine microalgae adapt to change in light quality and can be exploited for biofuel feedstock development.