Photoinactivation and long-term photosensitivity of diploid and disomic strains of the smut fungusUstilago violacea

Diploid and disomic strains ofUstilago violacea, containing high cellular concentrations of cytochrome c and various cellular concentrations of carotenes, were subjected to high levels of radiation from incandescent, fluorescent, and UV sources to characterize inactivation kinetics. Additionally, these strains were grown under high and low levels of radiation from the fluorescent and incandescent sources, to characterize their long-term resistance to photoinactivation by visible light with different spectral qualities. Survival kinetics in response to incandescent radiation were characterized by either an exponential decay for the low carotene strain, or an initial loss of viability, with recovery and a subsequent gradual loss of viability for the strains containing high carotene concentrations. Long-term colony survival was suppressed at the high incandescent level for the low carotene strain only. Long-term incandescent exposure did not affect the other strains. Survival kinetics in response to fluorescent and UV radiation were characterized as exponential decays for all strains, however, the exposure time leading to 90% loss in viability LD₉₀ was quantitatively related to carotene content in the form of a power function, where LD₉₀ increased as the total carotene content per cell. We also determined that the ratio of colored carotenes to cytochrome c in the cells was quantitatively related to the rate of viability loss during UV exposure. In all cases, including the long-term fluorescent experiment, the strains containing higher concentrations of carotenes were more resistant to destruction by the different radiation sources.     

Photosensitivity of pigmented and nonpigmented strains ofUstilago violacea

White, pink, pumpkin, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities in the presence and absence of the endogenous photosensitizer toluidine blue (TB). The white strain 15.10 and the cytochrome-c-accumulating pink strain 1.C429 were completely lacking carotenes and were the most sensitive to visible light under all conditions. The phytoene-containing white strain 2.C419, the carotene-containing pink strains AB278a-1, 1.C421, and 2.C425, and pumpkin strain 2.C415 were more resistant to photodestruction in the presence and absence of TB than strains 15.10 and 1.C429. Consistently, the most light resistant strains were the high carotene, low cytochrome-c-accumulating orange, and yellow strains. The photosensitivity of strains ofU. violacea was related to the level of carotene, cytochrome c, the presence or absence of TB, and the age of the culture.     

The kinetics of photoinactivation and long-term photosensitivity of pigmented and nonpigmented strains of the smut fungusUstilago violacea

White, pink, pumpkin, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities to characterize the kinetics of photoinactivation. Additionally, these strains were grown at two light intensities to characterize their long-term resistance to photoinactivation. The orange strain 2.D37291S and yellow strain 1.C2y had the highest carotene contents and were the most light resistant in the kinetics and growth experiments. The pink strains 2.C425, AB278a-1, and 1.C421 accumulated cytochrome c as well as carotenes. These strains were slightly more photosensitive than the yellow or orange strains in the kinetics experiment but were much more sensitive in the growth experiment. The phytoene-containing white strain 2.C419, which contains a small amount of cytochrome c, had a high level of resistance to light in the kinetics and growth experiments. The carotene-less strains were most sensitive in the kinetics experiment but not in the growth experiment. The overall photosensitivity ofU. violacea strains is related to the carotene and cytochrome c contents.     

Induction and Characterization of UV Resistant Calothrix braunii

A UV resistant mutant of Calothrix braunii has been isolated after repeated exposure to UV-C (254 nm) radiation. LD₅₀ for wild type against UV-B was 1.74 hand 100% lethality was achieved after 3.5 h. Whereas, UV resistant mutant showed LD₅₀ at 3.33 h and loss of complete survival after 5 h exposure. The growth rate of mutant was about 29 per cent greater than that of the wild type. The photosynthetic pigments, chlorophyll a and phycoerythrin were stimulated by 36.2 and 41.2 per cent, respectively over wild type. There were no differences in nitrogenase activity nitrate reductase activity, extracellular ammonia production and of plasmid DNA.     

Chromoplasts ultrastructure and estimated carotene content in root secondary phloem of different carrot varieties

There have been few studies on quantifying carotenoid accumulation in carrots, and none have taken the comparative approach. The abundance and distribution of carotenes in carrot roots of three varieties, white, orange, and high carotene mass (HCM) were compared using light and transmission electron microscopy (TEM). Light microscopy has indicated that, in all three varieties, carotenes were most abundant in the secondary phloem and this area was selected for further TEM analysis. While carotenes were extracted during the fixation process for TEM, the high-pressure freezing technique we employed preserved the spaces (CS) left behind by the extracted carotene crystals. Chromoplasts from the HCM variety contained significantly (P < 0.05) more CS than chromoplasts from the orange variety. Chromoplasts from the white variety had few or no CS. There was no significant difference between the HCM and orange varieties in the number of chromoplasts per unit area, but the white variety had significantly (P < 0.05) fewer chromoplasts than the other two varieties. A large number of starch-filled amyloplasts was observed in secondary phloem of the white variety but these were not found in the other two varieties. The results from this comparative approach clearly define the subcellular localization of carotenoids in carrot roots and suggest that while the HCM genotype was selectively bred for increased carotene content, this selection did not lead to increased numbers of carotene-containing chromoplasts but rather greater accumulation of carotene per chromoplast. Furthermore, the results confirm that roots of the white carrot variety retain residual amounts of carotene.     

Observations on Photosystem II mutants of Scenedesmus: Pigments and proteinaceous components of the chloroplasts

Nine mutants of the green alga, Scenedesmus obliquus, which are blocked in the Photosystem II portion of photosynthesis were analyzed for possible deletion or alteration of (1) various components of the photosynthetic electron transport system, (2) of chloroplast lipids, (3) of total chlorophyll or of the chlorophyll a/chlorophyllb ratio, and (4) of their content of carotenes and carotenoids. No changes in content or activity of ferredoxin, ferredoxin-NADP⁺ reductase, plastocyanin, cytochrome c-552, and the membrane-bound b-type or c-type cytochromes were observed. The most consistent differences noted between the mutant strains and the wild-type strain were in the molar ratio of chlorophyll/plastoquinone A, the total chlorophyll content, and a decreased content of - and β-carotene with a concomitant increase of carotenoids. The loss of Photosystem II activity in these mutant strains, as observed either with whole cells or with isolated chloroplast fragments, may be accounted for by their decreased content of plastoquinone A. Their decreased chlorophyll content and altered carotene/xanthophyll ratio also suggests possible alteration of chloroplast membrances resulting in increased internal oxidation of the photosynthetic pigments.     

Effects of Visible and Ultraviolet Light on Carotene-deficient Mutants of Crypthecodinium cohnii

Synopsis.
The ability of carotenoids to protect a heterotrophic dinoflagellate, Crypthecodinium cohnii , from photodynamic damage by sunlight in the presence of an exogenous dye was demonstrated. Wild-type C. cohnii and 2 carotcnoid-deficient mutants were plated on agar-solidified media and exposed to natural sunlight. The wild-type strain, which synthesizes γ–carotene and β–carotene, had the lowest mortality. Mutant car 17 which accumulates mainly §–carotene had an intermediate mortality rate while the albino mutant, car 3, which contains only phytoene, lost viability most rapidly. Wild-type cells treated with diphenylaminc, a carotenogenic inhibitor, were killed at the same rate as mutant car 3. The survival rate of mutants on exposure to sunlight was dependent upon the chromophore length of the accumulated carotenoids. The 3 strains showed no difference in rate of mortality when exposed to ultraviolet light. Protection from sunlight by accumulation of carotenes may be an important ecological factor for this species whose natural habitat is tidepools.     

Acclimation of tropical tree seedlings to excessive light in simulated tree-fall gaps

Acclimation to periodic high‐light stress was studied in tree seedlings from a neotropical forest. Seedlings of several pioneer and late‐succession species were cultivated under simulated tree‐fall gap conditions; they were placed under frames covered with shade cloth with apertures of different widths that permitted defined periods of daily leaf exposure to direct sunlight. During direct sun exposure, all plants exhibited a marked reversible decline in potential photosystem II (PSII) efficiency, determined by means of the ratio of variable to maximum Chl a fluorescence (F_v/F_m). The decline in F_v/F_m under full sunlight was much stronger in late‐succession than in pioneer species. For each gap size, all species exhibited a similar degree of de‐epoxidation of violaxanthin in direct sunlight and similar pool sizes of xanthophyll cycle pigments. Pool sizes increased with increasing gap size. Pioneer plants possessed high levels of β‐carotene that also increased with gap size, whereas α‐carotene decreased. In contrast to late‐succession plants, pioneer plants were capable of adjusting their Chl a/b ratio to a high value in wide gaps. The content of extractable UV‐B‐absorbing compounds was highest in the plants acclimated to large gaps and did not depend on the successional status of the plants. The results demonstrate a better performance of pioneer species under high‐light conditions as compared with late‐succession plants, manifested by reduced photoinhibition of PSII in pioneer species. This was not related to increased pool size and turnover of xanthophyll cycle pigments, nor to higher contents of UV‐B‐absorbing substances. High β‐carotene levels and increased Chl a/b ratios, i.e. reduced size of the Chl a and b binding antennae, may contribute to photoprotection in pioneer species.     

Lack of protection by carotenes against gamma-radiation damage in Phycomyces

Carotenes could protect cells from radiation damage by chemically quenching the free radicals and the activated chemical species originated by the exposure. We tested this hypothesis with strains of the zygomycete Phycomyces blakesleeanus that contained different carotenes (phytoene, lycopene, β-carotene) or different concentrations of β-carotene. Pairs of strains were cultured together, exposed to a maximum of 73 Gy γ-radiation from a ⁶⁰Co source, and allowed to recover and grow further together on limited resources. Irradiation did not affect the relative abundance of each strain in the resulting spore crop. Thus, carotenes did not protect the fungal cells against γ-radiation and did not influence their recovery from damage caused by the exposure. Received: 12 October 1995 / Accepted in revised form: 18 March 1996     

Neonatal phenobarbital administration results in increased cytochrome P450-dependent monooxygenase activity in adult male and female rats

The effects of neonatal exposure to phenobarbital during the first five days after birth on the enzymatic activity of the adult male and female rat liver P₄₅₀-dependent monooxygenase system were investigated. Although liver weight per 100 grams of body weight and total hepatic microsomal protein content were not altered in adult rats treated neonatally with phenobarbital, both sexes did show significant increases in cytochrome P₄₅₀ content, cytochrome P₄₅₀ reductase activity, cytochrome c reductase activity, ethoxycoumarin-O-deethylase activity and in the activity of a specific glucuronyl-transferase. Several of these activities were increased to a larger extent in the females, suggesting that females may be more sensitive to this phenomenon.     

Macromolecular Physiology of Plastids

The composition and amount of carotenoid pigments were determined in etiolated seedling leaves of 6 barley (Hordeum vulgare L.) mutants, comprising 1 xantha and 5 tigrina mutants. All mutants had on a mole basis approximately the same content of carotenoids as the wild type. The mutants xan-u²¹, tig-n³², and tig-³³ contained significantly higher amounts of carotenes than the wild type, ranging from 32 to 68% of the total carotenoid content as compared to the 4–8% found in the wild type. In the mutants tig-b²³ and tig-o³⁴, only a slight increase in the amount of carotenes was notable. The carotene content and composition in tig-d¹² was indistinguishable from that of the wild type. The carotenes extracted from xan-u²¹, tig-b²³, tig-n³², tig-³³, and tig-o³⁴ were characterized by adsorption chromatography and spectrophotometry. Mutant xan-u²¹ is in the dark blocked in β-carotene synthesis, and accumulates the aliphatic polyenes: phytofluene, proneurosporene, poly-cis-lycopenes, neo-lycopene and lycopene. The other four mutants synthesize β-carotene, but accumulate in addition various higher saturated carotenes, the main components being ζ-carotene in tig-b²³, a lycopenic pigment in tig-n³² and tig-³³, and lycopene in tig-o³⁴. Accumulation of higher saturated carotenes appears correlated with specific aberrations of the membrane structure in plastids. The regulation of carotene and protochlorophyllide syntheses in etioplasts are closely linked as shown by the single gene mutants which affect both pathways. However, several mutants have been identified which cause defects in protochlorophyllide synthesis only.     

Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation

Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone is not a sufficient measure for potential impact on biocontrol efficacy as other characters such as virulence may be severely affected even when viability remains high.     

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.     

Investigations into the effects of processing on the retention of the carotenoid fractions of Leucaena leucocephala during storage,and the effects of processing on mimosine concentration

Leucaena leucocephala leaves were subjected to several drying treatments followed by pelleting and/or inclusion of ethoxyquin as antioxidant and analysed monthly to determine rates of loss of carotenes and xanthophylls. Rapid sun drying yielded leucaena leaf meal with carotene and xanthophyll concentrations of 446 and 865 mg/kg, respectively, but considerable losses occurred during drying at 60 and 145°C, and as a result of blanching in hot water or ferrous sulphate solution. Loss rates of carotenes during storage were in the range 19–40 mg/kg per month, and of xanthophylls, 29–53 mg/kg per month. Carotenoids in sun-dried leaf meal were most stable during storage. Pelleting or inclusion of ethoxyquin did not reduce carotenoid loss rates below those of unpelleted leaf meal.The mimosine content of untreated sun-dried leucaena leaf meal was 3.2%, but the concentration decreased to 2.5 and 1.8% on drying at 60 and 145°C, respectively. The concentrations of mimosine in sun-dried leafmeals of water-blanched and ferrous sulphate-blanched leucaena were 2.0 and 2.1%, respectively. Treatments causing maximum losses of mimosine were those which brought about maximum losses of carotenoids.     

Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

Effects of Ultraviolet and X-Ray Radiation on in vitro Cultivated Cells of Haplopappus gracilis

A cell strain of Haplopappus gracills was used for investigations of the effects of UV (2537 A) and X-ray irradiation. Mitotic inhibition and killing after UV exposure were studied. A survival curve of UV treated and then plated cells is presented. The LD₅₀ seems to be about 2000 erg. mm^?2 under the experimental conditions used. All types of chromosome aberrations are induced by UV irradiation, but the frequency is relatively low at doses which do not completely inhibit cell division. A mutant strain of chromosome type is isolated after UV treatment and then plating. Mitotic inhibition and killing after X-ray treatment were studied. A survival curve is presented and the LD₅₀ under the culture conditions used seems to be about 2000 R. The frequency of chromosome aberrations induced by X-rays is highly increased by aeration during X-ray treatment which indicates that some degree of cell anoxia exists in a cell suspension. There arr indications that chromosome aherrations may not cause growth inhibition to such an extent as is usually believed.     

Mutants and Intersexual Heterokaryons of Blakeslea trispora for Production of β-Carotene and Lycopene

The industrial production of β-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of β-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N′-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having β-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of β-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of β-carotene per g (dry mass). β-Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.     

Major QTL for carrot color are positionally associated with carotenoid biosynthetic genes and interact epistatically in a domesticated × wild carrot cross

We performed QTL analyses for pigment content on a carotenoid biosynthesis function map based on progeny of a wild white carrot (QAL) which accumulates no pigments × domesticated orange carrot (B493), one of the richest sources of carotenoid pigments—mainly provitamin A α- and β- carotenes. Two major interacting loci, Y and Y ₂ on linkage groups 2 and 5, respectively, control much variation for carotenoid accumulation in carrot roots. They are associated with carotenoid biosynthetic genes zeaxanthin epoxidase and carotene hydroxylase and carotenoid dioxygenase gene family members as positional candidate genes. Dominant Y allele inhibits carotenoid accumulation. When Y is homozygous recessive, carotenoids that accumulate are either only xanthophylls in Y ₂ __ plants, or both carotenes and xanthophylls, in y ₂ y ₂ plants. These two genes played a major role in carrot domestication and account for the significant role that modern carrot plays in vitamin A nutrition.     

Poly-cis carotene pathway in the Scenedesmus mutant C-6D

The mutation of Scenedesmus obliquus strain C-6D is expressed in the dark only. Under these conditions, this strain synthesizes acyclic poly-cis carotenes. Cyclic carotenoids like -carotene or xanthophylls are absent. In the light the normal cyclic carotenes and xanthophylls are synthesized in the all-trans configuration. The poly-cis carotencs present in dark cultures have been analysed and quantitated. It could be shown that these poly-cis carotenes are identical with the poly-cis carotenes synthesized by the tomato mutant Lycopersicon esculentum var. Tangella. The poly-cis pathways, however, are different regulated in the two organisms. The tomato mutant accumulates prolycopene as the major carotene, whereas the mutant C-6D accumulates mainly pro--carotene. Furthermore, the mutation in Tangella is constitutive in light in contrast to Scenedesmus C-6D. Besides that, Scenedesmus C-6D synthesizes a further cis-carotene isomer of phytofluene as well as of -carotene. The configuration of these carotenes still have to be elucidated. The occurrence of this poly-cis carotene biosynthetic pathway by a mutation of only one enzyme, the phytoene desaturase which, however, is only expressed in darkness under heterotrophic conditions, is discussed.     

The Effect of γ-Radiation and Desiccation on the Viability of the Soil Bacteria Isolated from the Alienated Zone around the Chernobyl Nuclear Power Plant

Methylobacterium extorquens, M. mesophilicum, and Bacillus subtilis strains were found to be resistant to -radiation, irrespective of whether they were isolated from the alienated zone around the Chernobyl Nuclear Power Plant or outside this zone. The LD₉₀ of Methylobacterium and B. subtilis strains with respect to -radiation was 2.0–3.4 and 3.7–4.4 kGy, respectively, whereas their LD_99.99 values were 4.5–6.9 and more than 10 kGy, respectively. The high threshold levels of -radiation for Methylobacterium and B. subtilis imply the efficient functioning of DNA repair systems in these bacteria. Unlike Bacillus polymyxa cells, the cells of M. extorquens, M. mesophilicum, and B. subtilis were also resistant to desiccation. Pseudomonas sp., Nocardiasp., and nocardioform actinomycetes were sensitive to both -radiation and desiccation. Similar results were obtained when the bacteria studied were exposed to hydrogen peroxide and ultraviolet radiation. The results obtained indicate that the bacteria that are resistant to -radiation are also resistant to desiccation, UV radiation, and hydrogen peroxide. The possibility of using common laboratory tests (such as the determination of bacterial resistance to UV light and desiccation) for the evaluation of bacterial resistance to -radiation is discussed.