Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems

In the present study we have optimized the concentration of the bleaching herbicide norflurazon to obtain Dunaliella bardawil cells able to accumulate phytoene without losing viability. The highest concentration of phytoene 10.4 g/gChl was obtained for a concentration of norflurazon of 10 microg/mL. Norflurazon-treated Dunaliella bardawil cells are able to accumulate high concentrations of phytoene if the carotenogenic pathway is stimulated, but the lack of colored carotenoids make these cells particularly sensitive to high light intensities and to UVB radiation, so other stimuli, such as nitrogen starvation, have to be used to force the accumulation of phytoene. Detailed time-course evolution of the carotenoids lutein, violaxanthin, zeaxanthin, phytene and beta-carotene and the photosynthetic pigment chlorophyll was followed upon transfer of Dunaliella bardawil cells to nitrogen starvation in presence and absence of norflurazon. The combined use of the carotenogenic pathway inhibitor norflurazon and biphasic aqueous/organic systems to force the excretion of phytoene into the culture medium has been investigated. Cells cultured in the biphasic system were viable and able to produce phytoene during 3 days. Futhermore the productivity increased from 0.14 g/gChl . h in the aqueous culture to 0.18 g/gChl . h in the biphasic system. About 15% of the total phytoene produced by Dunaliella bardawil was excreted and immediately partionated into the organic phase. The concentration of phytoene in the decane phase was 2.05 g/gChl after 72 h, this means that about 47 g of phytoene per litre of culture were in the organic phase.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phqtoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low & carotene and in the nor-urazon-treated phytoene-rich cells. These obseruations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil portects the cells against injury by excessive irradiation.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.     

Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase

Light-stimulated carotenoid biosynthesis associated with the transformation of etioplasts to chloroplasts was investigated after dark-grown maize (Zea mays) seedlings were transferred into light. These studies focused on the enzymes of the pathway to detect those enzyme activities that were stimulated in the light and thus that were responsible for increased biosynthesis of carotenoids. In preliminary experiments, norflurazon, an inhibitor of phytoene desaturase, was used to prevent phytoene being further metabolized to carotenoids. Light-dependent stimulation of phytoene accumulation indicated that the light-regulated steps are located in the pathway leading to phytoene synthesis. The use of the 14C- labeled precursors mevalonic acid, isopentenyl pyrophosphate, and farnesyl pyrophosphate pointed to increased activity of an enzyme involved in the biosynthetic steps between isopentenyl pyrophosphate and farnesyl pyrophosphate. Determination of the activities of all five enzymes of the pathway involved in the sequence from mevalonic acid to phytoene revealed that the only enzyme activity stimulated by light was isopentenyl pyrophosphate isomerase. Over a 3-h period of illumination, this enzyme activity, like carotenoid biosynthesis, was stimulated 2.8-fold.     

达草灭对橙色大白菜愈伤组织中类胡萝卜素含量的影响

依据橙色大白菜的愈伤组织呈现橙色,而普通大白菜的愈伤组织呈现浅黄色这一现象,以橙色大白菜‘金冠1号’和普通大白菜‘秦白2号’培养的愈伤组织为材料,在培养基中添加0．1mmol．L-1的八氢番茄红素脱氢酶抑制剂达草灭分别处理5、10、20和40d。用HPLC法测定处理后的愈伤组织中各种类胡萝卜素含量的结果表明：‘秦白2号’愈伤组织中以β-胡萝卜紊为主,‘金冠1号’愈伤纽织中的类胡萝卜以番茄红素和β-胡萝卜素为主,且类胡萝卜素总含量比‘秦白2号’高出10．4倍;随着达草灭处理时间的延长,愈伤组织的颜色逐渐变白,其中番茄红素和β-胡萝卜素的含量逐渐下降,而八氢番茄红素的含量则逐渐升高,至处理40d时,两品种的愈伤组织中积累的八氢番茄红索差异不明显。据此,推论橙色大白菜中类胡萝卜素的积累并不是由于类胡萝卜素生物合成能力的提高引起的。     

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.Abbreviations BSA bovine serum albumin - D1 32 kDa Q_B-binding protein - EDTA ethylenediaminetetraacetic acid - LHCII light-harvesting complex II - PMSF phenylmethylsulfonyl fluoride - PS photosystem - tricine N-[tris(hydroxymethyl)methyl] glycine     

beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.     

Abstracts of papers read at the Winter Meeting, 1962

The effect of a range of inhibitors on the carotenoid biosynthetic pathway of the microalga Haematococcus pluvialis has been studied during normal growth and during the induction of astaxanthin synthesis. Diflufenican and norflurazon had similar effects and resulted in the almost complete inhibition of secondary carotenoid synthesis together with a build up of the acyclic carotenoid precursor, phytoene. In contrast, the inhibitor CPTA blocked cyclisation of lycopene and was seen to act differentially on the β- and ?-cyclases. Both diphenylamine and 1-aminobenzotriazole had the effect of blocking the synthesis of astaxanthin and the other secondary carotenoids by preventing the introduction of oxygen functions. As a direct result treated cells accumulated large levels of β-carotene instead. Selective use of inhibitors of carotenogenesis demonstrated that the accumulated lycopene and β-carotene could act as a precursor for astaxanthin synthesis.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta IV. Effect of Light on the Production of Ergosterol and Phytoene and on the Composition of Carotenoid Pigments

The effect of light on the production of ergosterol and phytoeneand on the composition of carotenoids in Rhodotorula minutawas studied to determine which part of the pathway of carotenoidsynthesis regulated by light. The ergosterol content in the cells was in the range of 3.4–3.6mg/g dry cells regardless of the presence or absence of illuminationand the light intensity. The phytoene production in the cellswas markedly stimulated by light and was dependent on the lightintensity according to the amount of carotenoid pigments produced.In addition, the ratio of phytoene to carotenoid was in therange of 0.36–0.44, regardless of the presence or absenceof illumination and the light intensity. The fact that the ratio of carotenoid fractionated on the basisof the functional group involved in each carotenoid to the totalamount of carotenoid was almost constant regardless of the lightintensity suggested that the composition of the carotenoidssynthesized in the cells is not affected by light. It was deduced from these results that light induced the productionof enzyme(s) required for phytoene biosynthesis in Rhodotorulaminuta. (Received November 7, 1981; Accepted March 19, 1982)     

Responses to bleaching herbicides by leaf chloroplasts of maize plants grown at different temperatures.

The effects of growth temperature on chloroplast responses to norflurazon and amitrole, two herbicides inhibiting carotenogenesis, at phytoene desaturation and lycopene cyclization, respectively, were studied in leaves of maize plants grown at 20 degrees C and 30 degrees C in light. At the lower temperature both chemicals caused severe photo-oxidative damage to chloroplasts. In organelles of norflurazon-treated leaves neither carotenoids nor chlorophylls were detectable and the thylakoid system was dismantled. In organelles of amitrole-treated leaves lycopene was accumulated, but small quantities of beta-carotene and xanthophylls were also produced. Moreover, some chlorophyll and a few inner membranes still persisted, although these latter were disarranged, lacking essential protein components and devoid of photosynthetic function. The increase in plant growth temperature to 30 degrees C did not change the norflurazon effects on carotenoid synthesis and the photo-oxidative damage suffered by chloroplasts. By contrast, in organelles of amitrole-treated leaves a large increase in photoprotective carotenoid biosynthesis occurred, with a consequent recovery of chlorophyll content, ultrastructural organization and thylakoid composition and functionality. This suggests that thermo-modulated steps could exist in the carotenogenic pathway, between the points inhibited by the two herbicides. Moreover it shows that, unlike C(3) species, C(4) species, such as maize, can express a strong tolerance to herbicides like amitrole, when supplied to plants growing at their optimum temperature conditions.     

Altered turnover of β-carotene and Chl a in Arabidopsis leaves treated with lincomycin or norflurazon

Interactions between β-carotene (β-C) and Chl a turnover were investigated in relation to photoinhibition and D1 protein turnover in mature leaves of Arabidopsis (Arabidopsis thaliana) by 1?CO? pulse-chase labeling. Following a 2 h treatment of leaves with water, lincomycin (Linco; an inhibitor of chloroplast protein synthesis) or norflurazon (NF; an inhibitor of carotenoid biosynthesis at phytoene desaturation) in the dark, 1?CO? was applied to the leaves for 30 min under control light (CL; 130 μmol photons m?2 s?1) conditions, followed by exposure to either CL or high light (HL; 1,100 μmol photons m?2 s?1) in ambient CO? for up to 6 h. Under both light conditions, 1?C incorporation was strongly decreased for Chl a and moderately suppressed for β-C in Linco-treated leaves, showing a marked decline of PSII efficiency (F(v)/F(m)) and β-C content compared with water-treated leaves. Partial inhibition of carotenoid biosynthesis by NF caused no or only a minor decrease in F(v)/F(m) and Chl a turnover under both conditions, while the β-C content significantly declined and high 1?C labeling was found for phytoene, the substrate of phytoene desaturase. Together, the results suggest coordinated turnover of Chl a and D1, but somewhat different regulation for β-C turnover, in Arabidopsis leaves. Inhibition of carotenoid biosynthesis by NF may initially enhance metabolic flux in the pathway upstream of phytoene, presumably compensating for short supply of β-C. Our observations are also in line with the notion that HL-induced accumulation of xanthophylls may involve a precursor pool which is distinct from that for β-C turnover.     

Regulation of carotenoid biosynthesis during tomato development.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.     

A possible structural role for carotenoids and carotenoid precursors in etioplasts

Seedlings of wheat ( Triticum aestivum L.) were grown in darkness in different concentrations of the herbicide SAN-9789, an inhibitor of carotenoid synthesis. The ultrastructural appearance of etioplasts, containing different amounts of carotenoids, was compared to the contents of carotenoids and carotenoid precursors (phytoene and phytofluene). A correlation was found between the presence of carotenoids and the presence of partitions between prothylakoids. As the plants were grown in darkness, this correlation is interpreted as the result of a structural role of the carotenoids. The presence of the herbicide SAN-9789 resulted in an increase in size and a change from osmiophilic to non-osmiophilic plastoglobuli. This change in plastoglobuli was neither correlated to the increase in phytoene or phytofluene, nor to the decrease in carotenoids.     

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Studies on the role of carotenoid pigments in a chemoheterotrophic bacterium,corynebacterium poinsettiae

Summary The hypothesis that colored carotenoids can protect chemoheterotrophic microorganisms from damage by visible light has been investigated. Corynebacterium poinsettiae, a bacterium that forms the three carotenoid pigments lycoxanthin, cryptoxanthin and spirilloxanthin, was used as test organism. Non-pigmented cells, in which the normal carotenoids were largely replaced by the colorless C₄₀ polyene, phytoene, were obtained by two methods: isolation of a mutant with a block in carotenoid synthesis; and cultivation of the parent strain in the presence of diphenylamine, a specific chemical inhibitor of carotenoid synthesis.Comparative studies of the effects of visible light on dye-sensitized pigmented and non-pigmented cells showed that non-pigmented cells can be rapidly killed by exposures which are without effect on pigmented cells. Both physiological and genetic suppression of pigment synthesis produce photosensitivity. The non-pigmented mutant is killed by ultraviolet light at the same rate as the pigmented parent strain, indicating that the acquired photosensitivity of the former is specific for visible light.Herrn Prof. Dr. A. Rippel zum 70. Geburtstag gewidmet.     

Production of phytoene, a carotenoid, and induction of connexin 26 in transgenic mice carrying the phytoene synthase gene crtB

Carotenoids have been recognized as chemopreventive agents against human diseases, such as cancer and cardiovascular disease. Mammalians utilize carotenoids supplied from their food since they are unable to perform the de novo synthesis of carotenoids. We previously created mammalian cultured cells producing phytoene, a type of carotenoid, and showed that these cells acquired resistance against oxidative stress and oncogenic transformation. In the present study, we established a transgenic mouse line, carrying the crtB gene encoding phytoene synthase, which could produce phytoene endogenously. It was found that connexin 26 was induced in these phytoene-producing mice. Since it is known that carotenoids enhance gap junctional communication by inducing the expression of connexin genes, the present data suggest that the induction of connexin 26 in phytoene-producing mice may play a role in controlling cell-to-cell communication. Phytoene-producing mice provide a useful system in which to investigate the in vivo function of the carotenoid phytoene.