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Background
Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage.
Results
Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance.
Conclusions
We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.
Microalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed.
Background
Flower longevity is closely related to pollen dispersal and reproductive success in all plants, as well as the commercial value of ornamental plants. Mutants that display variation in flower longevity are useful tools for understanding the mechanisms underlying this trait. Heavy-ion beam irradiation has great potential to improve flower shapes and colors; however, few studies are available on the mutation of flower senescence in leguminous plants.
Results
A mutant ( C416 ) exhibiting blossom duration eight times longer than that of the wild type (WT) was isolated in Lotus japonicus derived from carbon ion beam irradiation. Genetic assays supported that the delayed flower senescence of C416 was a dominant trait controlled by a single gene, which was located between 4,616,611 Mb and 5,331,876 Mb on chromosome III. By using a sorting strategy of multi-sample parallel genome sequencing, candidate genes were narrowed to the gene CUFF.40834, which exhibited high identity to ethylene receptor 1 in other model plants. A physiological assay demonstrated that C416 was insensitive to ethylene precursor. Furthermore, the dynamic changes of phytohormone regulatory network in petals at different developmental stages was compared by using RNA-seq. In brief, the ethylene, jasmonic acid (JA), and salicylic acid (SA) signaling pathways were negatively regulated in C416 , whereas the brassinosteroid (BR) and cytokinin signaling pathways were positively regulated, and auxin exhibited dual effects on flower senescence in Lotus japonicus . The abscisic acid (ABA) signaling pathway is positively regulated in C416 .
Conclusion
So far, C416 might be the first reported mutant carrying a mutation in an endogenous ethylene-related gene in Lotus japonicus , rather than through the introduction of exogenous genes by transgenic techniques. A schematic of the flower senescence of Lotus japonicus from the perspective of the phytohormone regulatory network was provided based on transcriptome profiling of petals at different developmental stages. This study is informative for elucidating the molecular mechanism of delayed flower senescence in C416 , and lays a foundation for candidate flower senescence gene identification in Lotus japonicus . It also provides another perspective for the improvement of flower longevity in legume plants by heavy-ion beam.
Xanthophyll cycle‐related non‐photochemical quenching, which is present in most photoautotrophs, allows dissipating excess light energy. Xanthophyll cycle‐related NPQ depends principally on xanthophyll cycle pigments composition and their effective involvement in non‐photochemical quenching. Xanthophyll cycle‐related NPQ is tightly controlled by environmental conditions in a species/strain specific manner. These features are especially relevant in microalgae living in a complex and highly variable environment. The goal of this study was to perform a comparative assessment of non‐photochemical quenching ecophysiologies across microalgal taxa in order to underline specific involvement of non‐photochemical quenching in growth adaptations and strategies. We used both published results and data acquired in our laboratory to understand the relationships between growth conditions (irradiance, temperature and nutrient availability), xanthophyll cycle composition and xanthophyll cycle pigments quenching efficiency in microalgae from various taxa. We found that in diadinoxanthin‐containing species, the xanthophyll cycle pigment pool is controlled by energy pressure in all species. At any given energy pressure, however, the diatoxanthin content is higher in diatoms than in other diadinoxanthin‐containing species. XC pigments quenching efficiency is species‐specific and decreases with acclimation to higher irradiances. We found a clear link between the natural light environment of species/ecotypes and quenching efficiency amplitude. The presence of diatoxanthin or zeaxanthin at steady state in all species examined at moderate and high irradiances suggests that cells maintain a light‐harvesting capacity in excess to cope with potential decrease in light intensity.
Background: Harnessing the halotolerant characteristics of microalgae provides a viable alternative for sustainable biomass and triacylglyceride (TAG) production. Scenedesmus sp. IITRIND2 is a fast growing fresh water microalga that has the capability to thrive in high saline environments. To understand the microalga’s adaptability, we studied its physiological and metabolic flexibility by studying differential protein, metabolite and lipid expression profiles using metabolomics, proteomics, real-time polymerase chain reaction, and lipidomics under high salinity conditions.
Results: On exposure to salinity, the microalga rewired its cellular reserves and ultrastructure, restricted the ions channels, and modulated its surface potential along with secretion of extrapolysaccharide to maintain homeostasis and resolve the cellular damage. The algal-omics studies suggested a well-organized salinity-driven metabolic adjust- ment by the microalga starting from increasing the negatively charged lipids, up regulation of proline and sugars accumulation, followed by direction of carbon and energy flux towards TAG synthesis. Furthermore, the omics studies indicated both de-novo and lipid cycling pathways at work for increasing the overall TAG accumulation inside the microalgal cells.
Conclusion: The salt response observed here is unique and is different from the well-known halotolerant microalga; Dunaliella salina, implying diversity in algal response with species. Based on the integrated algal-omics studies, four potential genetic targets belonging to two different metabolic pathways (salt tolerance and lipid production) were identified, which can be further tested in non-halotolerant algal strains.
Microalgae are capable of producing sustainable bioproducts and biofuels by using carbon dioxide or other carbon substances in various cultivation modes. It is of great significance to exploit microalgae for the economical viability of biofuels and the revenues from high-value bioproducts. However, the industrial performance of microalgae is still challenged with potential conflict between cost of microalgae cultivation and revenues from them, which is mainly ascribed to the lack of comprehensive understanding of carbon metabolism and energy conversion. In this review, we provide an overview of the recent advances in carbon and energy fluxes of light-dependent reaction, Calvin–Benson–Bassham cycle, tricarboxylic acid cycle, glycolysis pathway and processes of product biosynthesis in microalgae, with focus on the increased photosynthetic and carbon efficiencies. Recent strategies for the enhanced production of bioproducts and biofuels from microalgae are discussed in detail. Approaches to alter microbial physiology by controlling light, nutrient and other environmental conditions have the advantages of increasing biomass concentration and product yield through the efficient carbon conversion. Engineering strategies by regulating carbon partitioning and energy route are capable of improving the efficiencies of photosynthesis and carbon conversion, which consequently realize high-value biomass. The coordination of carbon and energy fluxes is emerging as the potential strategy to increase efficiency of carbon fixation and product biosynthesis. To achieve more desirable high-value products, coordination of multi-stage cultivation with engineering and stress-based strategies occupies significant positions in a long term.
Cement plants account for significant emissions of CO2 and other pollutants into the atmosphere. As a means for its mitigation, we tested the effect of a cement industry-based flue gas simulation (FGS — 18% CO2, 9% O2, 300 ppm NO2, 140 ppm SO2) on the green alga, Chlorella sorokiniana. Culture pH, cell density, cell viability and productivity, specific growth rates, photosynthetic performance, and biochemical composition were monitored. The treatments consisted of different FGS volumes (0.1, 0.3, 0.8, 1.5, 6, and 48 L day−1) that were applied in a series of laboratory-scale semi-continuous batch cultures under controlled conditions. Controls were exposed to 18% CO2 enriched air. Cell density showed that C. sorokiniana was able to grow in all treatments, but compared to the controls, low pH (~ 5.0) caused by 48 L FGS day−1 led to 27% decrease in specific growth rate. Increasing FGS exposure decreased maximum and operational quantum yields obtained by pulse amplitude modulated fluorometry, while photochemical quenching remained constant (~ 0.93). The α and rETRmax parameters calculated from rapid light curves decreased with increasing FGS exposure. Total proteins and carbohydrates (per cell basis) increased after 6 and 48 L FGS day−1, which can be advantageous for biotechnological applications, but cell productivity (cells L−1 day−1) decreased. Despite the effects in physiology, C. sorokiniana could withstand a pH range of 6.0–5.0 imposed by 48 L FGS day−1. Overall, C. sorokiniana can be considered a robust species in flue gas bioremediation.
Hypothesis The multiple stressors, in different combinations, may impact differently upon seed quality, and low-level doses of radiation may enhance synergistic or antagonistic effects.
Results During 1991–2014 we investigated the quality of the dandelion (Taraxacum officinale s.l.) seed progeny growing under low-level radiation exposure at the East-Ural Radioactive Trace (EURT) area (result of the Kyshtym accident, Russia), and in plants from areas exposed to background radiation. The viability of the dandelion seed progeny was assessed according to chronic radiation exposure, accounting for the variability of weather conditions among years. Environmental factors (temperature, precipitation, and their ratio in different months) can modify the radiobiological effects. We found a wide range of possible responses to multiple stressors: inhibition, stimulation, and indifferent effects in different seasons.
Conclusion The intraspecific variability of the quality of dandelion seed progeny was greatly increased under conditions of low doses of chronic irradiation. Temperature was the most significant factor for seed progeny formation in the EURT zone. Whereas the sums of precipitation and ratios of precipitation to temperature dominantly affected organisms from the background population.
A large proportion of mutants with altered pigment features have been obtained via exposure to heavy-ion beams, a technique that is efficient for trait improvement in the breeding of plants and algae. However, little is known about the underlying mechanisms by which the photosynthetic pigments are altered by heavy-ion irradiation. In our study, the photosynthetic characteristics of progenies from carbon-ion irradiated Selenastrum capricornutum were investigated. Five progenies deficient in chlorophyll a were isolated after carbon-ion exposure. Photosynthetic characteristics, photoprotection capacity and gene expression of the light-harvesting complex in these progenies were further characterized by the measurement of chlorophyll fluorescence parameters (Fv/Fm, ФPSII, NPQ, ETR), the de-epoxidation state of the xanthophyll cycle, the amount of lutein and quantitative real-time PCR. High maximum quantum yield of photosystem II at day 10 and high thermal dissipation ability were observed in progenies #23 and #37 under normal culture condition. Progenies #18, #19 and #20 showed stronger resistance against high levels of light steps than the control group (612-1077 μmol photons m -2 s -1, p< 0.05). The progenies #20 and #23 exhibited strong photoprotection by thermal dissipation and quenching of 3Chl* after 24 h of high light treatment. The mRNA levels of Lhcb5, Lhcbm5 and Lhcbm1 of the light-harvesting complex revealed markedly differential expression in the five progenies irradiated by carbon-ion beams. This work indicates that photosynthetic efficiency, photoprotection ability and the expression of light-harvesting antennae in unicellular green algae can be markedly influenced by irradiation. To our knowledge, this is the first report on changes in the photosynthetic pigments of green algae after treatment with carbon-ion beams.
Soil salinity severely threatens land use capability and crop yields worldwide. An analysis of the molecular mechanisms of salt tolerance in halophytes will contribute to the development of salt-tolerant crops. In this study, a combination of physiological characteristics and iTRAQ-based proteomic approaches was conducted to investigate the molecular mechanisms underlying the salt response of suspension cell cultures of halophytic Halogeton glomeratus. These cells showed halophytic growth responses comparable to those of the whole plant. In total, 97 up-regulated proteins and 192 down-regulated proteins were identified as common to both 200 and 400 mM NaCl concentration treatments. Such salinity responsive proteins were mainly involved in energy, carbohydrate metabolism, stress defense, protein metabolism, signal transduction, cell growth, and cytoskeleton metabolism. Effective regulatory protein expression related to energy, stress defense, and carbohydrate metabolism play important roles in the salt-tolerance of H. glomeratus suspension cell cultures. However, known proteins regulating Na⁺ efflux from the cytoplasm and its compartmentalization into the vacuole did not change significantly under salinity stress suggesting our existing knowledge concerning Na⁺ extrusion and compartmentalization in halophytes needs to be evaluated further. Such data are discussed in the context of our current understandings of the mechanisms involved in the salinity response of the halophyte, H. glomeratus.
Purpose:
An experiment was carried out to determine the effect of low dose gamma radiation on germination, plant growth, nitrogen and carbon fixation and carbon flow and release characteristics of groundnut.
Materials and methods:
Dry seeds of groundnut variety Trombay groundnut 37A (TG 37A), a radio mutant type developed by Bhabha Atomic Research Centre (BARC), Mumbai, India, were subjected to the pre-sowing treatment of gamma radiation within low to high dose physiological range, i.e., 0.0, 0.0082, 0.0164. 0.0328, 0.0656, 0.1312, 5, 25, 100, 500 Gray (Gy) from a cobalt source ((60)Co). Observations were recorded for the radiation effect on percentage germination, vigour, gas exchange attributes such as photosynthetic rate, stomatal conductance and transpiration rate, chlorophyll content, root exudation in terms of (14)C release, vascular sap flow rate and activities of rate defining carbon and nitrogen assimilating enzymes such as ribulose-1,5-bisphosphate carboxylase (rubisco) and nitrate reductase (NR).
Results:
Seed germination was increased by 10-25% at the lower doses up to 5 Gy while the improvement in plant vigour in the same dose range was much higher (22-84%) than the unirradiated control. For radiation exposure above 5 Gy, a dose-dependent decline in germination and plant vigour was measured. No significant effect was observed on the photosynthesis at radiation exposure below 5 Gy but above 5 Gy dose there was a decline in the photosynthetic rate. Stomatal conductance and transpiration rate, however, were only inhibited at a high dose of 500 Gy. Leaf rubisco activity and NR activities remained unaffected at all the investigated doses of gamma irradiation. Mean root exudation and sap flow rate of the irradiated plants, irrespective of the dose, was reduced over the unirradiated control more so in a dose-dependent manner.
Conclusions:
Results indicated that a very low dose of gamma radiation, in centigray to gray range, did not pose any threat and in fact stimulated metabolic functions in such a way to aid growth and development of groundnut plants. It further showed that the radiation threshold for the gas exchange traits and rubisco activity, which ultimately determine the plant health and yield, were higher than compared to the other metabolic attributes and were well beyond 500 Gy and that the dose range above 500 Gy should be targeted to measure lethal effects of radiation on carbon assimilation attributes in leguminous crops, in general, and groundnut in particular.
Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR) blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR.
Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach.
IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1.
Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells.
This study was conducted to develop a high-efficiency strain of Spirogyra varians for the production of biomass by radiation breeding. The characteristics of wild-type and mutant S. varians were analyzed through phenomenological and proteomic observations. The results of our phenomenological observations of the S. varians mutant demonstrated increases in growth rate and content of chlorophyll a, b, and a + b; in particular, a significant threefold increase was observed in starch accumulation. Proteomic analysis to investigate the differences in expression between wild-type and mutant proteins identified 18 proteins with significantly different expressions. From the literature review, it was confirmed that the up-regulated proteins were mainly involved in photosynthesis, carbohydrate biosynthesis, and energy metabolism. These results suggest the possibility of algae development by radiation breeding for the production of biofuel.
Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.
Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse species, including algae, but a preferred mutant cannot be easily obtained without a suitable large-scale screening method. We devised a unique, convenient, and effective method for screening mutants of Haematococcus pluvialis to isolate a strain resistant to environmental stress with low white fluorescence, i.e., a robust strain. Haematococcus was irradiated with heavy-ion beams of carbon ions, argon ions, and iron ions at various doses, after which approximately 10,000 surviving colonies were inoculated into 96-well plates, cultured for approximately 2 weeks, and then left to dry in a refrigerator for 3–12 months without a lid. In these unattended 96-well plates, cells in approximately one-third of the wells died and became white, and the remaining wells were approximately evenly split between red and green. The robustness of wild-type and mutant strains isolated from red and green wells was compared under severe environmental-stress conditions (125 µmol photons m⁻² s⁻¹, continuous light period, 45 mM sodium acetate). In the wild-type strain, most cells died, and 93.9% of cells emitted white autofluorescence. In contrast, few G4 cells emitted white autofluorescence, indicating a survival rate of 91.8%. Strains with excellent carotenoid production, such as G7 and R1, showed greater robustness compared to wild-type strains.
Lutein is a xanthophyll carotenoid with remarkable applications in the healthcare sector due to its ocular-protective, anti-oxidative and anti-inflammatory activities. Currently, marigold flower petals are the only commercial source for lutein extraction, since chemical synthesis suffers from very low yields. Microalgae cultivation is a promising way for lutein production, as the lutein content of microbial biomass is higher compared to that of marigold petals. In addition, microalgae are shown to have a higher growth rate, higher photosynthetic efficiency, and carbon mitigation potential when compared to terrestrial plants, such as marigolds. For the realization of microalgae as a sustainable and alternative lutein source, crucial factors such as the microalgal strains, critical cultivation parameters (light intensity, nutritional status, metabolic mode), pretreatment, extraction, and purification of lutein from microalgal biomass need to be studied in great detail. In this context, this review summarized the recent research progress in lutein production from microalgae, including competent microalgal strains, cultivation systems, and subsequent biomass treatment technologies. The choice of a high yielding strain, along with effective photobioreactor design will enable maximal lutein production from microalgae in an economically viable manner. This review presents comprehensive information on microalgal cultivation and pretreatment which will enable researchers to design an optimal microalgae-based lutein production system.
Conventional wastewater treatment using activated sludge cannot efficiently eliminate nitrogen and phosphorus, thus engendering the risk of water eutrophication and ecosystem disruption. Fortunately, a new wastewater treatment process applying microalgae-bacteria consortia has attracted considerable interests due to its excellent performance of nutrients removal. Moreover, some bacteria facilitate the harvest of microalgal biomass through bio-flocculation. Additionally, while stimulating the functional bacteria, the improved biomass and enriched components also brighten bioenergy production from the perspective of practical applications. Thus, this review first summarizes the current development of nutrients removal and mutualistic interaction using microalgae-bacteria consortia. Then, advancements in bio-flocculation are completely described and the corresponding mechanisms are thoroughly revealed. Eventually, the recent advances of bioenergy production (i.e., biodiesel, biohydrogen, bioethanol, and bioelectricity) using microalgae-bacteria consortia are comprehensively discussed. Together, this review will provide the ongoing challenges and future developmental directions for better converting nitrogen and phosphorus wastewater into bioenergy using microalgae-bacteria consortia.
The chlorophyte microalga Chlorella sorokiniana was tested for the bioremediation of heavy metals pollution. It was cultured with different concentrations of Cu⁺², Cd⁺², As (III) and As (V), showing a significant inhibition on its growth at concentrations of 500 µM Cu⁺², 250 µM Cd⁺², 750 µM AsO3⁻³ and 5 mM AsO4⁻³ or higher. Moreover, the consumption of ammonium was also studied, showing significant differences for concentrations higher than 1 mM of Cu⁺² and As (III), and 5 mM of As (V). The determination of intracellular heavy metals concentration revealed that Chlorella sorokiniana is an outstanding Cd accumulator organism, able to accumulate 11,232 mg kg⁻¹ of Cd, and removing 65% of initial concentration of this heavy metal. Finally, antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and enzymes involved in the production of glutamate and cysteine, such as glutamine syntethase (GS), glutamate dehydrogenase (GDH), O-acetylserine (thiol) lyase (OASTL) and NAD-isocitrate dehydrogenase (NAD-IDH) were studied both at gene expression and enzymatic activity levels. These enzymes exhibited different grades of upregulation, especially in response to Cd and As stress. However, GS expression was downregulated when Chlorella sorokiniana was cultured in the presence of these heavy metals.
The differentially expressed proteins of mutated C. pyrenoidosa obtained by N⁺ ion implantation were isolated and identified using proteomics analysis method combining the isobaric tags for relative and absolute quantitation and liquid chromatography-tandem mass spectrometry techniques. The functions of these proteins were also investigated toward elucidating the fundamental mechanism by which ion implantation promotes the growth of C. pyrenoidosa and accumulation of lipids at the molecular level. In total, 17 differentially expressed proteins were identified in algae cells, including 3 up-regulated and 14 down-regulated proteins. Combining these results with the central carbon metabolism network of C. pyrenoidosa cells and the lipid metabolism pathway showed that N⁺ ion implantation can promote the synthesis of chlorophyll by up-regulating magnesium protoporphyrin IX methyltransferase to further improve the carbon sequestration efficiency of algae cells. These phenomena result in a greater production rate of organic material and therefore increased biomass. In addition, N+ ion implantation can enhance the glycolysis and pentose phosphate pathways by down-regulating fructose-bisphosphate aldolase and transketolase. These effects ultimately promote the accumulation of lipids by C. pyrenoidosa. These findings provide a rationalization of the mechanism by which ion implantation-induced mutagenesis promoting high lipid productivity in C. pyrenoidosa CVM at the proteomics level.
Parachlorella kessleri (formerly Chlorella kessleri) can accumulate high levels of both starch and lipids that can be used for the production of bioethanol and biodiesel, respectively. We mutagenized P. kessleri by heavy-ion beam irradiation and obtained a biotechnologically promising mutant strain, PK4, for further improvement of lipid productivity in comparison with wild type [1]. PK4 reached two times the optical density and accumulated 2.6 times as much starch in complete urea-phosphate (UP) media compared to tris-acetate-phosphate (TAP) media under laboratory conditions. Similarly to wild type, PK4 accumulated only negligible amounts of lipids in complete UP media. PK4 accumulated more lipids (WT: 1.17 g L−1, PK4: 1.75 g L−1) and accumulated lipids faster than WT after dilution of the UP media (WT; 0.22 g L−1 day−1, PK4; 0.43 g L−1 day−1). The productivity of PK4 was analyzed in mass culture using a 150-L thin-layer photo bioreactor housed in a temperature and lightuncontrolled glass greenhouse. The PBR consisted of two glass plates (each 6m long and 1m wide) at an inclination of 1.6%, arranged in a meandering path and connected with a trough. The culture was diluted four-fold with water on day 7 post-inoculation to ensure prompt nutrient limitation in the UP media. Lipid accumulation in PK4 was significantly induced so that it accumulated 66% of lipid per dry weight. Under 150-L mass cultivation conditions, PK4 showed high biomass productivity (0.82 g L−1 day−1) and high lipid productivity (0.59 g L−1 day−1). In addition, the whole genome of PK4 was sequenced to clarify the genetic variation upon heavy-ion-beam irradiation by comparison with the reference genome of WT. As a result, genetic differentiation of PK4 was found at three genes encoding endo-1,4-β-mannanase, an ATP/ADP transporter, and an elicitor response protein.
The aquatic environment is continuously exposed to ionizing radiation from both natural and anthropogenic sources, making the characterization of ecological and health risks associated with radiation of large importance. Microalgae represent the main source of biomass production in the aquatic ecosystem, thus becoming a highly relevant biological model to assess the impacts of gamma radiation. However, little information is available on the effects of gamma radiation on microalgal species, making environmental radioprotection of this group of species challenging. In this context, the present study aimed to improve the understanding of the effects and toxic mechanisms of gamma radiation in the unicellular green algae Chlamydomonas reinhardtii focusing on the activity of the photosynthetic apparatus and ROS formation. Algal cells were exposed to gamma radiation (0.49–1677 mGy/h) for 6 hrs and chlorophyll fluorescence parameters obtained by PAM fluorometry, while two fluorescent probes carboxy-H2DFFDA and DHR 123 were used for the quantification of ROS. The alterations seen in functional parameters of C. reinhardtii PSII after 6 hrs of exposure to gamma radiation showed modifications of PSII energy transfer associated with electron transport and energy dissipation pathways, especially at the higher dose rates used. Results also showed that gamma radiation induced ROS in a dose-dependent manner under both light and dark conditions. The observed decrease in photosynthetic efficiency seems to be connected to the formation of ROS and can potentially lead to oxidative stress and cellular damage in chloroplasts. To our knowledge, this is the first report on changes in several chlorophyll fluorescence parameters associated with photosynthetic performance and ROS formation in microalgae after exposure to gamma radiation.
The experimental results revealed that the 2.27-fold lipid yield was enhanced in nitrogen-depleted condition (226mg/L) when compared to nitrogen rich condition (99.33mg/L). Since specific growth rate (SGR) is the single most criteria to decide the biomass yield of microalgae, SGR was analyzed. SGR of 0.33day(-1) was observed with 0.47 doubling per day under nitrogen rich condition. However, low growth rate of 0.14day(-1) with a doubling of 0.20 per day was recorded under nitrogen depleted condition. In accordance with SGR, the dry biomass yield was ranged from 0.055±0.005 to 1.394±0.010g/L in the presence of nitrogen, indicating the essentiality of major nutrient in the growth medium. The enhanced lipid accumulation under nitrogen starved condition by Scenedesmusquadricauda was perhaps achieved by adopting adverse environmental condition and possible increment in cell size in terms of length/breadth ratio (1.11).
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Plants exposed to ionising radiation (IR) have to face direct and indirect (oxidative stress) deleterious effects whose intensity depends on the dose applied and led to differential genome regulation. Transcriptomic analyses were conducted with CATMA microarray technology on Arabidopsis thaliana plantlets, 2 and 26h after exposure to the IR doses 10Gy and 40Gy. 10Gy treatment seemed to enhance antioxidative compound biosynthetic pathways whereas the 40Gy dose up-regulated ROS-scavenging enzyme genes. Transcriptomic data also highlighted a differential regulation of chloroplast constituent genes depending on the IR dose, 10Gy stimulating and 40Gy down-regulating. This probable 40Gy decrease of photosynthesis could help for the limitation of ROS production and may be coupled with programmed cell death (PCD)/senescence phenomena. Comparisons with previous transcriptomic studies on plants exposed to a 100Gy dose revealed 60 dose-dependent up-regulated genes, including notably cell cycle checkpoints to allow DNA repairing phenomena. Furthermore, the alteration of some cellular structure related gene expression corroborated a probable mitotic arrest after 40Gy. Finally, numerous heat-shock protein and chaperonin genes, known to protect proteins against stress-dependent dysfunction, were up-regulated after IR exposure.
The present study describes the isolation of pigmentation mutants of Porphyra yezoensis Ueda induced by heavy-ion beam irradiation for the first time. The gametophytic blades were irradiated with 12C+6 ion beams within a dose range of 25–400 Gy. From the survival rate and cell growth of the irradiated blades, it is suggested that a dose of 150 Gy or less is suitable to induce mutation for the isolation of mutants of P. yezoensis. After irradiation, red, green and deep reddish brown-colored gametophytic blades developed from archeospores that were released from each of the mutated cell clusters of the respective different colors, and the red mutant strain (IBY-R1) and green mutant strain (IBY-G1) were established as a conchocelis colony in culture. Blades of the mutants were characterized by their growth and photosynthetic pigment contents compared with those of the wild-type. From these results, it is clear that heavy-ion beam mutagenesis will be an effective tool for genetic and breeding studies of Porphyra, and also for other algal research.
As a green-tide-forming macroalga, Ulva linza is distributed worldwide and therefore subject to various environmental stresses. The LHCSR (also known as LI818 in green
alga and LHCX in diatoms) protein is a stress-related member of the LHC family that plays an important role in photo-protective
mechanism, which has been only found in algae. In this study, we cloned full-length cDNA sequence encoding the LhcSR gene from U. linza and analyzed its expression in response to different temperature and illumination gradients. The results showed that high
light (HL) could enhance expression of LhcSR and that the expression level peaked at 3h under HL. Similarly, the expression of LhcSR could also be induced by low temperature (LT). However, the expression patterns of LhcSR were quite different in response to LT and HL treatment. Specifically, the maximum gene expression under LT was much higher
(11.8-fold) than under HL (5.4-fold) when compared to the expression under normal conditions. The upregulated expression of
LhcSR lasted for 12h under LT, but 3h under HL. These data suggest that the LhcSR gene is involved in photoprotection in U. linza, and the results suggest a stronger link to LT. In addition, the discrepancy in expression under HL and LT was consistent
with the ecological features of this alga, which only thrives during the cold season (featured as LT and low light).
Keywords
Ulva linza
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LhcSR–Full-length–Expression analysis–Green tide
The efficiency of pigment extraction forms the crux of the spectrophotometric analysis of chlorophyll a. The alcoholic solvents, methanol and ethanol, proved to be superior to acetone and acetone with DMSO. Homogenisation and sonication did not improve the extraction in the alcoholic solvents. Boiling at 100C had an adverse effect whereas complete extraction of the pigments was obtained at the solvents boiling point and allowing the samples to stand for 24 h in the dark.
Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Heavy-ion irradiation is a new method of mutation breeding to produce new cultivars. We established the application of this method in rice plants to obtain mutants. Rice seeds were irradiated by C or Ne ions (135MeV/u) with a LET (linear energy transfer) of 22.7 or 64.2 keV/microm, respectively. Chlorophyll-deficient mutants (CDM) segregated in M2 progeny were albino, pale-green, yellow or striped-leave phenotypes. The highest rate of CDM with C-ion irradiation, 7.31%, was obtained at 40 Gy among the doses examined. Ne-ion irradiation gave the highest rate, 11.6%, at 20 Gy. We used the RLGS (Restriction Landmark Genomic Scanning) method to analyze DNA deletion in an albino mutant genome. Not I-landmark RLGS profiles detected about 2000 spots in rice. We found that one of the polymorphic spots was strongly linked to the albino phenotypic mutant derived from deleting of a DNA fragment, and demonstrated the high ability to detect of polymorphic regions by the RLGS method.
A viewpoint: why chlorophyll a? Photosynthesis Research
- L O Björn
- G C Papageorgiou
- R E Blankenship
- G J P Research
Extraction and quantification of pigments from a marine microalga: a simple and reproducible method. Communicating Current Research and Educational Topics and Trends in Applied Microbiology Formatex
- M Henriques
- A Silva
- J Rocha
Chlorophyll fluorescence-a practical guide
- Maxwell