Modification in the ascorbate-glutathione cycle leads to a better acclimation to high light in the rose Bengal resistant mutant of Nannochloropsis oceanica.

Understanding how to adapt outdoor cultures of Nannochloropsis oceanica to high light (HL) is vital for boosting productivity. The N. oceanica RB2 mutant, obtained via ethyl methanesulfonate mutagenesis, was chosen for its tolerance to Rose Bengal (RB), a singlet oxygen (1O2) generator. Compared to the wild type (WT), the RB2 mutant showed higher resilience to excess light conditions. Analyzing the ascorbate-glutathione cycle (AGC), involving ascorbate peroxidases (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.8.1.7), in the RB2 mutant under HL stress provided valuable insights. At 250 µmol photon m-2 s-1 (HL), the WT strain displayed superoxide anion radicals (O2▪-) and hydrogen peroxide (H2O2) accumulation, increased lipid peroxidation, and cell death compared to normal light (NL) conditions (50 µmol photon m-2 s-1). The RB2 mutant didn't accumulate O2▪- and H2O2 after HL exposure, and exhibited increased APX, DHAR, and GR activities and transcript levels compared to WT and remained consistent after HL treatment. Although the RB2 mutant had a smaller ascorbate (AsA) pool than the WT, its ability to regenerate dehydroascorbate (DHA) increased post HL exposure, indicated by a higher AsA/DHA ratio. Additionally, under HL conditions, the RB2 mutant displayed an improved glutathione (GSH) regeneration rate (GSH/GSSG ratio) without changing the GSH pool size. Remarkably, H2O2 or menadione (a O2▪- donor) treatment induced cell death in the WT strain but not in the RB2 mutant. These findings emphasize the essential role of AGC in the RB2 mutant of Nannochloropsis in handling photo-oxidative stress.

Photoacclimation of photosystem II photochemistry induced by rose Bengal and methyl viologen in Nannochloropsis oceanica.

The photosynthetic apparatus is a major reactive oxygen species (ROS) proliferator, especially in high-light environments. The role of ROS in photoinhibition and photoacclimation mechanisms has been extensively explored, primarily in model plant species. However, little work has been performed on the topic in non-Archaeplastida organisms, such as the model heterokont species Nannochloropsis oceanica. To investigate the photoacclimation and damaging impact of singlet oxygen and superoxide anions on the photosynthetic apparatus of N. oceanica, we subjected cells to two doses of methyl viologen and rose bengal. Significant findings: Rose bengal (a singlet-oxygen photosensitizer) induced changes to the photosynthetic apparatus and PSII photochemistry mirroring high-light-acclimated cells, suggesting that singlet-oxygen signaling plays a role in the high-light acclimation of PSII. We further suggest that this singlet-oxygen pathway is mediated outside the plastid, given that rose bengal caused no detectable damage to the photosynthetic apparatus. Methyl viologen (a superoxide-anion sensitizer) induced an enhanced non-photochemical quenching response, similar to what occurs in high-light-acclimated cells. We propose that superoxide anions produced inside the plastid help regulate the high-light acclimation of photoprotective pathways.

Characterization of Nannochloropsis oceanica Rose Bengal Mutants Sheds Light on Acclimation Mechanisms to High Light When Grown in Low Temperature.

A barrier to realizing Nannochloropsis oceanica's potential for omega-3 eicosapentaenoic acid (EPA) production is the disparity between conditions that are optimal for growth and those that are optimal for EPA biomass content. A case in point is temperature: higher content of polyunsaturated fatty acid, and especially EPA, is observed in low-temperature (LT) environments, where growth rates are often inhibited. We hypothesized that mutant strains of N. oceanica resistant to the singlet-oxygen photosensitizer Rose Bengal (RB) would withstand the oxidative stress conditions that prevail in the combined stressful environment of high light (HL; 250 µmol photons m-2 s-1) and LT (18°C). This growth environment caused the wild-type (WT) strain to experience a spike in lipid peroxidation and an inability to proliferate, whereas growth and homeostatic reactive oxygen species levels were observed in the mutant strains. We suggest that the mutant strains' success in this environment can be attributed to their truncated photosystem II antennas and their increased ability to diffuse energy in those antennas as heat (non-photosynthetic quenching). As a result, the mutant strains produced upward of four times more EPA than the WT strain in this HL-LT environment. The major plastidial lipid monogalactosyldiacylglycerol was a likely target for oxidative damage, contributing to the photosynthetic inhibition of the WT strain. A mutation in the NO10G01010.1 gene, causing a subunit of the 2-oxoisovalerate dehydrogenase E1 protein to become non-functional, was determined to be the likely source of tolerance in the RB113 mutant strain.

Overcoming Intrinsic Restriction Enzyme Barriers Enhances Transformation Efficiency in Arthrospira platensis C1.

The development of a reliable genetic transformation system for Arthrospira platensis has been a long-term goal, mainly for those trying either to improve its performance in large-scale cultivation systems or to enhance its value as food and feed additives. However, so far, most of the attempts to develop such a transformation system have had limited success. In this study, an efficient and stable transformation system for A. platensis C1 was successfully developed. Based on electroporation and transposon techniques, exogenous DNA could be transferred to and stably maintained in the A. platensis C1 genome. Most strains of Arthrospira possess strong restriction barriers, hampering the development of a gene transfer system for this group of cyanobacteria. By using a type I restriction inhibitor and liposomes to protect the DNA from nuclease digestion, the transformation efficiency was significantly improved. The transformants were able to grow on a selective medium for more than eight passages, and the transformed DNA could be detected from the stable transformants. We propose that the intrinsic endonuclease enzymes, particularly the type I restriction enzyme, in A. platensis C1 play an important role in the transformation efficiency of this industrial important cyanobacterium.

Extremophilic micro-algae and their potential contribution in biotechnology.

Micro-algae have potential as sustainable sources of energy and products and alternative mode of agriculture. However, their mass cultivation is challenging due to low survival under harsh outdoor conditions and competition from other, undesired, species. Extremophilic micro-algae have a role to play by virtue of their ability to grow under acidic or alkaline pH, high temperature, light, CO2 level and metal concentration. In this review, we provide several examples of potential biotechnological applications of extremophilic micro-algae and the ranges of tolerated extremes. We also discuss the adaptive mechanisms of tolerance to these extremes. Analysis of phylogenetic relationship of the reported extremophiles suggests certain groups of the Kingdom Protista to be more tolerant to extremophilic conditions than other taxa. While extremophilic microalgae are beginning to be explored, much needs to be done in terms of the physiology, molecular biology, metabolic engineering and outdoor cultivation trials before their true potential is realized.

ACCLIMATION TO LOW TEMPERATURE OF TWO ARTHROSPIRA PLATENSIS (CYANOBACTERIA) STRAINS INVOLVES DOWN-REGULATION OF PSII AND IMPROVED RESISTANCE TO PHOTOINHIBITION(1).

This study aimed to compare the ability of two Arthrospira platensis (Nordst.) Gomont strains, M2 and Kenya, isolated from two different habitats, to acclimate to low temperature (15°C). Both strains had similar growth rates at 30°C, but once acclimated to low temperature, M2 showed a greater decline in growth (59% vs. 41% in the Kenya strain). We suggest that the Kenya strain acclimated better to low temperature by down-regulating its photosynthetic activity through (i) decreasing antenna size and thus reducing energy flux into the photosystems; (ii) decreasing reaction center density (RC/CSX ) and the performance index, thus decreasing the trapping probability and electron transport rate while maintaining electron transport probability for electron transport beyond QA (-) unchanged; (iii) increasing the energy dissipation flux. In contrast, the M2 strain showed no difference in antenna size and exhibited a much lower decrease in RC/CSX and a lower dissipation rate. Hence, the Kenya strain minimized potential damage on the acceptor side of PSII compared to the M2 cells. Furthermore, acclimation to low temperature was accompanied by an improved mechanism for handling excess energy resulting in an enhanced ability of the Kenya strain to rapidly repair damaged PSII RCs and withstand a high photon flux density (HPFD) stress; this finding might be defined as a cross-adaptation phenomenon. This study may provide a tool to identify strains suitable for outdoor mass-production in different regions characterized by different climate conditions.

Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid.

We have hypothesized that among algae of alpine environment there could be strains particularly rich in long chain polyunsaturated fatty acids (LC-PUFA). Indeed, the chlorophyte (Trebuxiophyceae) Parietochloris incisa isolated from Mt. Tateyama, Japan, was found to be the richest plant source of the pharmaceutically valuable LC-PUFA, arachidonic acid (AA, 20:4omega6). The alga is also extremely rich in triacylglycerols (TAG), which reaches 43% (of total fatty acids) in the logarithmic phase and up to 77% in the stationary phase. In contrast to most algae whose TAG are made of mainly saturated and monounsaturated fatty acids, TAG of P. incisa are the major lipid class where AA is deposited, reaching up to 47% in the stationary phase. Except for the presence of AA, the PUFA composition of the chloroplastic lipids resembled that of green algae, consisting predominantly of C(16) and C(18) PUFAs. The composition of the extrachloroplastic lipids is rare, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as diacylglyceryltrimethylhomoserine (DGTS). PC and PE are particularly rich in AA and are also the major depots of the presumed precursors of AA, l8:3omega6 and 20:3omega6, respectively.

Effects of salinity stress on photosystem II function in cyanobacterial Spirulina platensis cells.

The changes in PSII photochemistry in Spirulina platensis cells exposed to salinity stress (0-0.8 M NaCl) for 12 h were studied. Salinity stress induced a decrease in oxygen evolution activity, which correlated with the decrease in the quantum yield of PSII electron transport (PhiPSII). Phycocyanin content decreased significantly while chlorophyll content remained unchanged in salt-stressed cells. Salinity stress induced an increase in non-photochemical quenching (qN) and a decrease in photochemical quenching (qP). Analyses of the polyphasic fluorescence transients (OJIP) showed that with the increase in salt concentration, the fluorescence yield at the phases J, I and P declined sharply and the transient almost levelled off at salt concentration of 0.8 M NaCl. The effects of DCMU on the polyphasic rise of fluorescence transients decreased significantly. Salinity stress resulted in a decrease in the efficiency of electron transfer from QA- to QB. The slope at the origin of the relative variable fluorescence curves (dV/dto) and the relative variable fluorescence at phase J (VJ) increased in the absence of DCMU, but decreased in the presence of DCMU. The shape of the relative variable fluorescence transients in salt-stressed cells was comparable to that of the control cells incubated with DCMU. The results in this study suggest that salt stress inhibited the electron transport at both donor and acceptor sides of PSII, resulted in damage to phycobilisome and shifted the distribution of excitation energy in favour of PSI.

MIXOTROPHIC GROWTH MODIFIES THE RESPONSE OF SPIRULINA (ARTHROSPIRA) PLATENSIS (CYANOBACTERIA) CELLS TO LIGHT.

Spirulina (Arthrospira) platensis (Nordstedt) Geitler cells grown under mixotrophic conditions exhibit a modified response to light. The maximal photosynthetic rate and the light saturation value of mixotrophic cultures were higher than those of the photoautotrophic cultures. Dark respiration and light compensation point were also significantly higher in the mixotrophically grown cells. As expected, the mixotrophic cultures grew faster and achieved a higher biomass concentration than the photoautotrophic cultures. In contrast, the growth rate of the photoautotrophic cultures was more sensitive to light. The differences between the two cultures were also apparent in their responses to exposure to high photon flux density of 3000 µmol·m-2 ·s-1 . The light-dependent O2 evolution rate and the maximal efficiency of photosystem II photochemistry declined more rapidly in photoautotrophically grown than in mixotrophically grown cells as a result of exposure to high photon flux density. Although both cultures recovered from the high photon flux density stress, the mixotrophic culture recovered faster and to a higher extent. Based on the above results, growth of S. platensis with a fixed carbon source has a significant effect on photosynthetic activity.

Characterization of PSII photochemistry in salt-adapted cells of cyanobacterium Spirulina platensis.

The changes in pigment composition, photosynthesis and PSII photochemistry were investigated in cells of Spirulina platensis adapted to salt stress (<0.75 M NaCl). A decrease in the phycocyanine/chlorophyll and no significant change in the carotenoid/chlorophyll ratio were observed in salt-adapted cells. Salt stress inhibited the apparent quantum efficiency of photosynthesis and PSII activity while stimulating PSI activity and dark respiration significantly. Salt stress also resulted in a decrease in overall activity of the electron transport chain, which could not be restored by diphenylcarbazide, an artificial electron donor to the reaction centres of PSII. Measurements of the polyphasic fluorescence rise in fluorescence transients including phases O, J, I and P showed that salt stress had no effect on the fluorescence yield at phase O but decreased the fluorescence yield at phases J, I and P. Analyses of the JIP test developed from the polyphasic rise of fluorescence transients showed that salt stress led to a decrease in both the maximum quantum efficiency of PSII photochemistry and the maximum quantum efficiency of electron transport beyond the primary quinone electron acceptor. However, salt stress induced no significant changes in the probability of transporting an electron beyond QA , the trapping flux per PSII reaction centre, or the electron transport flux per PSII reaction centre. A theoretical analysis of fluorescence parameters indicated a decrease in the rate constant of excitation energy trapping by PSII reaction centres. In addition, salt stress induced an increase in the complementary area above the fluorescence induction curve in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting an increase in the proportion of closed PSII reaction centres in salt-adapted cells. Based on these results, it is suggested that modifications in PSII photochemistry in salt-adapted Spirulina cells maintained a high conversion efficiency of excitation energy, such that no significant change was observed in either the trapping flux or the electron transport flux per PSII reaction centre.