Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Possible Role of Cbr,an Algal Early-Light-Induced Protein,in Nonphotochemical Quenching of Chlorophyll Fluorescence

The unicellular green alga Dunaliella bardawil exhibits typical responses to excessive light when starved for sulfate under normal light (60 [mu]E m-2 s-1) but not under low light (14 [mu]E m-2 s-1). Algae were analyzed during several days of sulfate starvation for nonphotochemical quenching of chlorophyll fluorescence in the absence or presence of the uncouplers SF-6847 (SF) or carbonyl cyanide p- trifluoromethoxyphenyl hydrazone. Parallel analyses followed two light-stress responses: (a) violaxanthin conversion to zeaxanthin and (b) accumulation of Cbr, a protein analogous to plant early-light-induced proteins and implicated in zeaxanthin binding. In cells starved under normal light SF inhibited nonphotochemical quenching during the first 24 h, but not from 40 h onward. In cells starved under low light SF inhibited nonphotochemical quenching throughout the starvation period. Under normal light accumulation of zeaxanthin was nearly maximal by 24 h, but Cbr was fully induced only by 40h. Under low light zeaxanthin accumulated slowly but no Cbr was evident. These results suggest that during exposure to excessive light, the initial pH gradient-dependent, Cbr-independent mode of nonphotochemical quenching is modified to become less dependent on pH gradient and requires Cbr.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

Involvement of zeaxanthin and of the Cbr protein in the repair of photosystem II from photoinhibition in the green alga Dunaliella salina.

A light-sensitive and chlorophyll (Chl)-deficient mutant of the green alga Dunaliella salina (dcd1) showed an amplified response to irradiance stress compared to the wild-type. The mutant was yellow-green under low light (100 micromol photons m(-2) s(-1)) and yellow under high irradiance (2000 micromol photons m(-2) s(-1)). The mutant had lower levels of Chl, lower levels of light harvesting complex II, and a smaller Chl antenna size. The mutant contained proportionately greater amounts of photodamaged photosystem (PS) II reaction centers in its thylakoid membranes, suggesting a greater susceptibility to photoinhibition. This phenotype was more pronounced under high than low irradiance. The Cbr protein, known to accumulate when D. salina is exposed to irradiance stress, was pronouncedly expressed in the mutant even under low irradiance. This positively correlated with a higher zeaxanthin content in the mutant. Cbr protein accumulation, xanthophyll cycle de-epoxidation state, and fraction of photodamaged PSII reaction centers in the thylakoid membrane showed a linear dependence on the chloroplast 'photoinhibition index', suggesting a cause-and-effect relationship between photoinhibition, Cbr protein accumulation and xanthophyll cycle de-epoxidation state. These results raised the possibility of zeaxanthin and Cbr involvement in the PSII repair process through photoprotection of the partially disassembled, and presumably vulnerable, PSII core complexes from potentially irreversible photooxidative bleaching.     

DNA fingerprinting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world

Background

Dunaliella salina is the most important species of the genus for β-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.

Results

In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and β-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached β-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.

Conclusion

In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.     

Under low irradiation,the light regime modifies growth and metabolite production in various species of microalgae

The effects of continuous light exposure (24L:0D) and a 12 h:12 h light/dark regime (12L:12D) were compared on the growth and carotenoid, protein, sugar, lipid, and fatty acid contents in Chlorella vulgaris, Nannochloropsis sp., Isochrysis galbana, and Dunaliella salina cultured in a batchwise facility. These microalgae were grown axenically under a low photon flux density (PFD) of 27 μmol photons m^?2 s^?1. C. vulgaris, Nannochloropsis sp., and I. galbana exhibited the highest cell densities when cultured under 24L:0D, whereas D. salina grew better under the alternating light/dark regime. I. galbana accumulated high levels of proteins, sugars, and lipids and exhibited the highest carotenoid content under 24L:0D. Protein production was enhanced in C. vulgaris under 24L:0D. The highest total lipid content was recorded for D. salina, reaching 74.6 % of total proteins, sugars, and lipids in cells at the stationary phase when grown under 12L:12D. The light/dark regime at low PFD was sufficient to stimulate the accumulation of monounsaturated and polyunsaturated fatty acids in all four algae. Their levels, like those of saturated fatty acids, did not differ significantly under the two light regimes. D. salina was an important source of tetradecenoic acid 14:1(n-5). Nannochloropsis sp. produced a large amount of the essential eicosapentaenoic acid, which reached 20 % of total fatty acids under 12L:12D, while I. galbana exhibited the highest level of docosahexaenoic acid, which reached 21 % under both light regimes. This study demonstrated the feasibility of culturing microalgae under low PFD in order to produce large quantities of valuable metabolites, especially various lipids with neutraceutical value.     

The effect of light quality on leaf production and development of in vitro-cultured plants of Alternanthera brasiliana Kuntze

We investigated the influence of light quality on the leaf development of Alternanthera brasiliana Kuntze (Amaranthaceae) grown in vitro. Growth parameters including specific leaf mass, thickness, and leaf density were lowest in plants grown under red light. Blue light induced the largest number of leaves/plant, and the largest thickness and area of the leafblade. Green and red lights induced the smallest leaf areas. The thickness of the abaxial-face epidermis and spongy parenchyma of the plants was significantly reduced in plants grown under red light. The thickness of the palisade parenchyma and upper epidermis were significantly increased in plants grown under blue light, compared to the other fluorescent-light treatments. The specific spectral band also influenced the differentiation of mesophyll cells. In the dark and under red light, the mesophyll was homogenous; and in the dark and under green light, the leaves were more compact. Under blue light, the cells displayed the characteristic palisade morphology. The results showed that the increase of a specific parenchyma type was related to a specific spectral band. All spectral-quality treatments reduced the numbers of stomata and trichomes. The results for green light were in some respects similar to those for red light, and in other respects similar to those for blue light, probably because phytochromes and cryptochromes are green-light receptors. This study indicated that Alternanthera plants have strong morphological plasticity induced by light. The results suggest that high-quality Alternanthera can be achieved by culturing the plants in vitro under a combination of blue and red light.     

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.     

The Selectivity of Milking of Dunaliella salina

The process of the simultaneous production and extraction of carotenoids, milking, of Dunaliella salina was studied. We would like to know the selectivity of this process. Could all the carotenoids produced be extracted? And would it be possible to vary the profile of the produced carotenoids and, consequently, influence the type of carotenoids extracted? By using three different D. salina strains and three different stress conditions, we varied the profiles of the carotenoids produced. Between Dunaliella bardawil and D. salina 19/18, no remarkable differences were seen in the extraction profiles, although D. salina 19/18 seemed to be better extractable. D. salina 19/25 was not “milkable” at all. The milking process could only be called selective for secondary carotenoids in case gentle mixing was used. In aerated flat-panel photobioreactors, extraction was much better, but selectiveness decreased and also chlorophyll and primary carotenoids were extracted. This was possibly related to cell damage due to shear stress.     

Ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light

The ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta Thuret grown in red, blue and green light was studied both in ultrathin sections and in replicas of rapidly frozen cells. High activity of dictyosornes and mucilage sacs results in a dramatic decrease of the protoplasmic area and in thicker cell walls in red light in comparison with blue light and the control. There are numerous well‐formed phycobili‐somes in blue light, whereas not well‐formed ones are present in red and especially in green light. There are also many phycobilisomes in the intrapyrenoidal thylakoids in blue light, fewer in green light, but they are absent in red light and in the control. It seems that in red and especially in green light, the phycobilisomes have fewer rods than in blue light. In green light, chloroplasts bear numerous genophores in contrast to blue and red light. The spacings of neighboring parallel thylakoids are as follows: control 64.3 nm, blue light 90.6 nm, red light 41.3 nm, green light 43.7 nm. Due to the relatively small spacing of the neighboring parallel thylakoids in red (41.3 nm) and in green light (43.7 nm) and of the given height of phycobilisomes (35 nm), the alternate phycobilisomes attached to neighboring lamellae are forced to interdigitate. The density of phycobilisomes per square micrometer of thylakoid surface dramatically increases in blue light (800 μm^?2) in relation to red (250 μm^?2) and green light (180 μm^?2). The protoplasmic fracture face of the thylakoids reveals numerous, tightly packed, but randomly distributed particles. The particle size distribution is uniform in the two types of fracture faces, with an average diameter of about 11.5 nm. In blue light, both the phycobilisomes and exoplasmic face particles are organized into rows with a spacing of 60–70 nm. The results (changes: in the protoplasmic area; in the spacing of the thylakoids; in phycobilisome arrangement; in structure, shape and size of phycobilisomes; and in the accumulation of plastoglobuli), have shown that the monochromatic light (blue, red and green) brings about marked changes in the package effect and consequently in the efficiency of light absorption. In addition, the blue light contributes to the intense production of chlorophyll a, phycoerythrin, phycocyanin and soluble proteins, while intense production of polysaccharidic material is attributed to red light.     

Green light signaling and adaptive response

To a plant, the sun’s light is not exclusively energy for photosynthesis, it also provides a package of data about time and prevailing conditions. The plant’s surroundings may dampen or filter solar energies, altering spectral profiles of their light environment. Plants use this information to adjust form and physiology, tailoring gene expression to best match ambient conditions. Extensive literature exists on how blue, red and far-red light contribute to plant adaptive responses. A growing body of work identifies discrete effects of green light (500–565 nm) that also shape plant biology. Green light responses are known to be either mediated through, or independent of, the cryptochrome blue light receptors. Responses to green light share a general tendency to oppose blue- or red-light-induced responses, including stem growth rate inhibition, anthocyanin accumulation or chloroplast gene expression. Recent evidence demonstrates a role for green light in sensing a shaded environment, independent from far-red shade responses.     

Green light drives photosynthesis in mosses

Temperate forests are characterised by variable light quality (i.e. spectral composition of light) at or near the forest floor. These understory environments have a high concentration of green light, as red and blue light are preferentially absorbed by upper canopy leaves. Understory species may be well-adapted for using green light to drive photosynthesis. Angiosperms have been shown to use green light for photosynthesis, but this ability has not been demonstrated in shade-dwelling bryophytes. In this study, net photosynthetic rate (P_N) of three temperate understory species of moss (Dichodontium pellucidum (Hedw.) Schimp., Leucobryum albidum (Brid. ex P.Beauv) Lindb. and Amblystegium serpens (Hedw.) Schimp.) was measured under green, red?+?blue, and red?+?blue?+?green light to assess green light use efficiency. All three species were capable of photosynthesising beyond their respiratory demands using solely green light, with higher green light use efficiency measured in plants collected from areas with greater canopy cover, suggesting growth in a green light concentrated environment increases green light use efficiency. Each species was also collected from sites differing in their degree of canopy cover and grown under three light treatments (high light, low light, and green light). Photosynthetic efficiency (chlorophyll fluorescence), tissue nitrogen and carbon isotope concentrations were assessed after a short growth period. Growth conditions had little effect on leaf chemistry and monochromatic green light did not significantly degrade photosynthetic efficiency. This study provides the first evidence to date of positive net ‘green light photosynthesis’ in mosses.     

Acclimations to light quality on plant and leaf level affect the vulnerability of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) to water deficit

We investigated the influence of light quality on the vulnerability of pepper plants to water deficit. For this purpose plants were cultivated either under compact fluorescence lamps (CFL) or light-emitting diodes (LED) providing similar photon fluence rates (95 µmol m^?2 s^?1) but distinct light quality. CFL emit a wide-band spectrum with dominant peaks in the green and red spectral region, whereas LEDs offer narrow band spectra with dominant peaks at blue (445 nm) and red (665 nm) regions. After one-week acclimation to light conditions plants were exposed to water deficit by withholding irrigation; this period was followed by a one-week regeneration period and a second water deficit cycle. In general, plants grown under CFL suffered more from water deficit than plants grown under LED modules, as indicated by the impairment of the photosynthetic efficiency of PSII, resulting in less biomass accumulation compared to respective control plants. As affected by water shortage, plants grown under CFL had a stronger decrease in the electron transport rate (ETR) and more pronounced increase in heat dissipation (NPQ). The higher amount of blue light suppressed plant growth and biomass formation, and consequently reduced the water demand of plants grown under LEDs. Moreover, pepper plants exposed to high blue light underwent adjustments at chloroplast level (e.g., higher Chl a/Chl b ratio), increasing the photosynthetic performance under the LED spectrum. Differently than expected, stomatal conductance was comparable for water-deficit and control plants in both light conditions during the stress and recovery phases, indicating only minor adjustments at the stomatal level. Our results highlight the potential of the target-use of light quality to induce structural and functional acclimations improving plant performance under stress situations.     

The effect of light on the content of photosynthetically active pigments in species of the genus Chara

The photosynthetic pigments, chlorophyll a, b and carotenoids, were studied in three species of the Chara genus, as representatives of the phycobenthos, grown under white, red, green and blue light.The highest values of the photosynthetic pigments were noted in the three species of algae under green and yellow light, whereas the lowest occurred under red and blue light.     

Translocation in colored light

The translocation of ¹⁴C-photosynthate in detached blades of sugarcane was studied under illumination from red, green, blue, and cool-white fluorescent lamps; under far-red illumination from the sun, and from incandescent lamps; and in total darkness.

The percentage of basipetal translocation and the accumulation against the concentration gradient were stimulated by light from the red or blue lamps more than by green or cool-white fluorescent illumination.

Basipetal translocation took place equally well in red light lacking blue irradiation and in blue light. Since the action spectrum for light-induced change in viscosity is a typical blue-type spectrum, the effect of light upon translocation is not due merely to changes in the physicochemical properties of protoplasm.

Basipetal translocation took place in red light lacking blue irradiation better than in cool-white fluorescent light, which may suggest a red stimulation of translocation.

Illumination in the far-red region of the spectrum did not support basipetal translocation but acted like total darkness.

Because of the wide emission characteristics of the fluorescent lamps employed, it is impossible to decide whether a chlorophyll-like system or some other pigment is involved in the light stimulation of phototranslocation.

Whatever the activating wavelength and whatever the pigment system involved, these results show that the phototranslocation of sucrose in the phloem is influenced by the quality of illumination.

     

Exposure of Dunaliella salina to Low Temperature Mimics the High Light-Induced Accumulation of Carotenoids and the Carotenoid Binding Protein (Cbr)

We report that growth of Dunaliella salina at either 13°C/150µmol m^–2s^–1 or 30°C/2,500 µmol m^–2s^–1 results in the accumulation of comparable levels ofcarotenoids and the zeaxanthin-binding protein, Cbr. We concludethat carotenoid and Cbr abundance in this green alga respondto changes in PSII ‘excitation pressure’ ratherthan to high light per se. (Received September 19, 1996; Accepted November 20, 1996)     

Effects of light quality on the chloroplastic ultrastructure and photosynthetic characteristics of cucumber seedlings

To understand how light quality influences plant photosynthesis, we investigated chloroplastic ultrastructure, chlorophyll fluorescence and photosynthetic parameters, Rubisco and chlorophyll content and photosynthesis-related genes expression in cucumber seedlings exposed to different light qualities: white, red, blue, yellow and green lights with the same photosynthetic photon flux density of 100 μmol m^?2 s^?1. The results revealed that plant growth, CO₂ assimilation rate and chlorophyll content were significantly reduced in the seedlings grown under red, blue, yellow and green lights as compared with those grown under white light, but each monochromatic light played its special role in regulating plant morphogenesis and photosynthesis. Seedling leaves were thickened and slightly curled; Rubisco biosynthesis, expression of the rca, rbcS and rbcL, the maximal photochemical efficiency of PSII (Fv/Fm) and quantum yield of PSII electron transport (Ф_PSII) were all increased in seedlings grown under blue light as compared with those grown under white light. Furthermore, the photosynthetic rate of seedlings grown under blue light was significantly increased, and leaf number and chlorophyll content of seedlings grown under red light were increased as compared with those exposed to other monochromatic lights. On the contrary, the seedlings grown under yellow and green lights were dwarf with the new leaves etiolated. Moreover, photosynthesis, Rubisco biosynthesis and relative gene expression were greatly decreased in seedlings grown under yellow and green light, but chloroplast structural features were less influenced. Interestingly, the Fv/Fm, Ф_PSII value and chlorophyll content of the seedlings grown under green light were much higher than those grown under yellow light.