Betacyanin and antioxidant system in tolerance to salt stress in Alternanthera philoxeroides. Peters, Eugenia J. Bolacel Braga. Received: April, Approved: February, Salinity is a limiting factor fot growth and development, since it affects several physiological processes in plants. This trial aimed to assess the levels of photosynthetic pigments, betacyanins, lipid peroxidation and activity of antioxidant enzymes in Alternanthera philoxeroides under salt stress.
Plants originated from an in vitro culture were acclimatized and irrigated with sodium chloride 0, and mM for 30 d. Higher levels of betacyanin were observed on stems, when compared to the leaves, in the highest salt concentrations.
On the leaves, there was a significant increase of lipid peroxidation and superoxide dismutase activity, whereas roots showed an increase of the enzymes catalase and ascorbate peroxidase. Salt stress caused greater degradation in the photosynthetic pigments, increment of betacyanin synthesis in stems, and damage to the cell membranes of the leaves.
However, the increase of antioxidant enzymes activity stimulated the protective system against oxidative stress. It is concluded that Alternanthera philoxeroides Mart. Key words: Amaranthaceae, alligator weed, sodium chloride, photosynthetic pigments, antioxidant enzymes. Las plantas se originaron de un cultivo in vitro y se aclimataron e irrigaron con cloruro de sodio 0, y mM durante 30 d.
Se concluye que las plantas de Alternanthera philoxeroides Mart. Salinity is one of the most important factors among abiotic stress limiting plant growth and yield, because it affects almost all physiological and biochemical processes, reducing the yield Flowers, High salt concentrations induce water stress hydric deficit , causing cellular and ionic imbalance, due to the toxicity of these ions, and promoting osmotic stress Mandhania et al.
Plants, in order to protect their cell membranes and organelles from the damaging effects of these radicals, activate an antioxidant defense system consisting of superoxide dismutase SOD-EC 1. But if the complete reduction does not occur under conditions of increased production, the result can be a state of stress leading to oxidation of lipids, proteins and DNA.
These events can trigger inactivation of cellular components, accelerating the process of cell death Buckner et al. In this sense, the antioxidant defense system plays a key role towards the acquisition of tolerance. Leaf pigments such as chlorophylls and carotenoids can also be used as indicative parameters of stress, when plants are cultivated under high salt concentrations.
Depending on the species under study, cultivar, exposure time and salt concentration, such stress may affect the absorption of nutrients that constitute the chlorophyll molecule and the formation of other photosynthetic pigments, therefore hampering photosynthesis itself Santos, The species Alternanthera philoxeroides Mart.
These plants are composed of flavonoids, saponins and betalains; betalains are natural N-heterocyclic pigments soluble in water that is divided into two structural groups: the betaxanthins yellow and betacyanins red to red purple. Betacyanins are widely used as an additive to food and drugs, being an nontoxic natural dye Azeredo, ; Volp et al.
Moreover, betalains play important roles in stress physiology, acting as ROS resultant from abiotic stresses detoxifiers, as it was observed in Beta vulgaris subsp. Native to South America this species is mainly found in pioneer formations, "restingas", usually in recent sandy terrains Blum, It is classified as a perennial species that grows abundantly in different ecosystems, from aquatic environments to those extremely dry, such as dunes Gao et al.
Given the above, this study aimed to investigate the effect of salinity on chlorophyll contents, total carotenoids, betacyanins, lipid peroxidation and antioxidant enzymes in plants of A. The plants were kept 10 d in a moist chamber with irrigation microsprinkler ; then plants were removed from the humid chamber and transferred to plastic pots 2 L capacity containing the same substrate and maintained in a greenhouse.
The plants received mL of Hoagland complete nutritive solution Hoagland and Arnon, every two days during 10 d; afterwards the application of mL of NaCl per pot was initiated, at concentrations of 0, and mM, at intervals of four consecutive days, interspersed with water, during 30 d and then leaves, stems and roots were collected for biochemical analyses.
The chlorophylls Chl a, Chl b and total - Chlt and carotenoids were evaluated using the method described by Lichtenthaler with some modifications. Betacyanin values were expressed as a function of the amarantine content. The reaction was stopped using rapid cooling on ice and the readings carried out in a spectrophotometer at nm and nm. The leaves used for this analysis were formed after the onset of the induction treatment.
The SOD activity was assessed by the ability of the enzyme to inhibit the photo-reduction of the nitroblue tetrazolium NBT Giannopolitis and Ries, in a reaction medium comprising mM potassium phosphate pH 7. CAT activity was determined according to the protocol described by Azevedo et al. The plant extract was added prior to reading in the spectrophotometer. The APX activity was quantified following Nakano and Asada , by monitoring the ascorbate oxidation rates at nm during 2 min.
For analyses of betacyanins the stem and leaf were used and for the enzymes analysis, roots and leaf. Leaves removed from each treatment NaCl concentration were used for the analyses of pigments. There were three repetitions and four plants per plot were the experimental unit.
The contents of pigments showed significant differences between treatment NaCl concentrations and control Table 1. Salt stress significantly reduced the levels of chlorophyll; however, among the saline treatments and mM NaCl , no difference was found. This response indicates that in the presence of high salt concentration plants are unable to synthesis chlorophyll or alternatively that a greater degradation of such pigments occurs.
Salt stress also negatively affected the carotenoids content, with a significant reduction in the levels of these pigments in plants subjected to mM NaCl when compared to control. The plant's ability to grow and adapt to different environments is related to its reproductive efficiency, which is associated to the levels of chlorophyll.
The content of chlorophylls in the leaves is used to estimate the photosynthetic potential of the plants, through its direct connection with the absorption and transfer of light energy and growth and adaptation to various environments Almeida et al.
In plants, oxidative stress and signs of senescence include loss of chlorophyll, which lead to a progressive reduction in photosynthetic capacity Thompson et al. The smaller content of chlorophylls in plants subjected to salt stress can be explained by a decrease in its synthesis or higher degradation of these pigments, because during the process of degradation of chlorophyll Chl b is converted to the Chl a Fang et al.
Panda and Khan also observed a reduction in chlorophyll and carotenoids in Vigna radiata plants after 24 h of salt stress. Santos noted that Helianthus annuus L. Besides, the enzyme chlorophyllase is stimulated to synthesize chlorophyll in the first days of moderate stress Santos, However, such increased activity is strongly inhibited by high salt concentration.
Salt stress also leads to an increase of free radicals in the chloroplasts and consequently to the destruction of chlorophyll molecules, resulting in reduced photosynthetic ability and growth. In this situation, the chlorophylls must be degraded as soon as possible to prevent cellular damage due to its photodynamic action Takamiya et al.
If the chlorophyll degradation does not occur efficiently, it can be followed by an increase in the ROS production to an extent that the antioxidant system is no longer capable detoxify it, thus requiring the activation of other antioxidant systems Dolatabadian et al.
Carotenoids are pigments related to cell protection against photo-oxidative damage, especially the xanthophylls cycle violaxanthin, antheraxanthin and zeaxanthin Garcia-Plazaola and Becerril, ; Mittler, Provitamin A carotenoids can be turned into vitamin A retinol in the intestine or liver.
Vitamin A is an important component to human health. It helps maintain eye health, healthy mucus membranes and immunity. Alpha-carotene, beta-carotene and beta-cryptoxanthin are provitamin A carotenoids; lutein, zeaxanthin and lycopene are not. Lutein and zeaxanthin are associated primarily with eye health. Studies often do not separate lutein and zeaxanthin because they are the only carotenoids found in the retina. Scientists seem to know more about lutein, and supplements typically contain much more lutein than zeaxanthin.
It has been shown to reduce the incidence of cataract lens opacity and light sensitivity if consumed in adequate quantities on a daily basis. Premkumar noted that lutein could also be good for the heart. When lutein is in the blood, it can have an antioxidant effect on cholesterol, thereby preventing cholesterol from building up in the arteries and clogging them.
A study published in Circulation found that participants who added lutein supplements to their diets had less arterial wall thickening than those who did not. Good sources of lutein and zeaxanthin include kale, spinach, turnip greens, summer squash, pumpkin, paprika, yellow-fleshed fruits and avocado, said Premkumar.
Lutein is also available through enriched eggs. A study published in the Journal of Nutrition found that lutein from enriched eggs was absorbed better than lutein from spinach or supplements. Beta-cryptoxanthin is a xanthophyll carotenoid that is also provitamin A. It can be a source of vitamin A, but it produces half as much as beta-carotene. Premkumar listed papaya, mango and oranges as good sources of it.
Beta-cryptoxanthin is typically found in yellow foods, such as corn and bell peppers, and is present in yellow-colored dairy products, such as egg yolks and butter. Some studies have shown that beta-cryptoxanthin may be effective in preventing lung cancer.
In an analysis of several studies from North America and Europe, published in Cancer Epidemiology, Biomarkers and Prevention , researchers found that participants who consumed the most beta-cryptoxanthin had a 24 percent lower chance of developing lung cancer than those with the lowest consumption.
In a large-scale study conducted in the Netherlands and also published in Cancer Epidemiology, Biomarkers and Prevention , researchers found that though all carotenoids were measured for their relationship to lung cancer risk, only beta-cryptoxanthin, lutein and zeaxanthin were associated with reduced cancer risk. Beta-cryptoxanthin may be helpful in reducing the risk of inflammatory polyarthritis, which includes rheumatoid arthritis.
Similarly, Gu et al. An alternative to decrease the industrial operation costs during the synthesis of microbial carotenoids is the use of agro-industrial wastes as substrates. Carotenoid pigments synthesis in yeasts starts at the late logarithmic phase continuing in the stationary phase [ 82 , 91 , 92 ], the presence of a reliable carbon source is important for carotenoid biosynthesis during the stationary phase.
Yeasts can synthesize carotenoids when cultivated in commercial medium, containing various refined carbon sources, such as glucose [ 34 , 66 , 70 , 74 , 79 , 86 , 87 , 92 — 96 ], xylose [ 97 ], cellobiose [ 96 ], sucrose [ 98 , 99 ], glycerol [ 79 ] and sorbitol [ 91 , 96 ], nevertheless these type of medium represents high costs. As a result, studies on carotenogenesis have led to find out low-cost substrate as alternative to reduce production costs. Therefore, there have been a growing interest in the use of natural substrates as carbon sources such as grape juice [ 65 , ]; grape must [ , ]; peat extract and peat hydrolyzate [ 65 , 82 , , ]; date juice of Yucca filifera [ 81 ], hydrolyzed mustard waste isolates [ ], hemicellulosic hydrolyzates of eucalyptus globules wood [ , ], hydrolyzed mung bean waste flour [ ], sugar cane juice [ 80 , , ], sugar cane and sugar-beet molasses [ 42 , 67 , ], corn syrup [ 45 , 77 ], corn hydrolyzate [ , ], milk whey [ 67 , 71 , 72 , 75 , 78 ].
These by-products from industrial processes are pollutants to the environmental and their treatment represents high costs. In recent years, raw materials and agro-industrial wastes origin have been proposed as low-cost alternative carbohydrate sources.
On this regard, chicken feathers and sweet potato have been introduced as nitrogen and carbon sources [ ], respectively to produce carotenoid, and also minimizing environmental and energetic problems related to their disposal [ ]. Many investigations have been performed to diminish the costs and optimize the carotenoid production; and factors such as carbon and nitrogen source are very important to consider on the selection of agro-industrial waste as substrates [ ].
A wide spread natural substrate is milk whey; it contains lactose, proteins and minerals, principally. Biological wastewater treatment technologies can assist in safe disposal of whey within environmental specifications, but these are expensive [ ] becoming an attractive low-cost substrate for microbial production of carotenoids.
Nasrabadi and Razavi [ ] reported a carotenoid production of a lactose positive mutant strain R. Aksu and Eren [ 68 ] reported that the addition of cottonseed oil to the R. Table 1 presents the most recent agro-industrial residues used as substrate to microbial carotenoid production, and the carotenoid quantities obtained. Production of carotenoid with agro-industrial wastes depends on the kind carbon and nitrogen source, minerals and other components, as well as the quantities of each one.
The composition of these materials is very important to define the preparation of culture media to improve the carotenogenesis on microorganisms. Nowadays, the importance of biotechnological processes has increased due to benefits that they provide such as high yields, low costs and less waste disposals. These benefits are dependent on nutrients and the culture conditions such as inoculum size, pH, aeration, agitation, and others.
To design a biotechnological process, many factors must be taken in consideration such as bioreactor design, raw materials, the microorganism or enzyme, type of fermentation batch, feed-batch or continuous. These features play a very important role to achieve the desired yields of a target metabolite [ ].
The configuration and volume of the bioreactor is an important factor to consider in the microbial production of carotenoids. Several investigations using yeast for the production of microbial carotenoids in stirred tank bioreactors have been published Table 2. A fully functional bioreactor offers advantages such as perfect integration of several components, ensuring that cultures will reach the desired productivity of microbial pigments or other microbial compounds, through an efficient and rigorous control of some parameters such as temperature, agitation, aeration, pH and dissolved oxygen among others.
Figure 2 illustrates a scheme of a general biotechnological process where the bioreactor is the central part of the process.
General scheme of biotechnological processes for carotenoids production. Industrial applications of bioprocess to produce microbial carotenoids generally employ genetically modified strains, being hyper-producing strains that had been manipulated to maximize carotenoids production.
Among the several strategies to increase overall yield in a microbial carotenoids bioprocess, metabolic engineering for the yeast carotenoids producers in an interesting research area. Selection of the appropriate substrate. The raw materials utilized might or not be pretreated depending on the fermentative capacity of the microorganism and the type of enzymes produced.
The bioreactor configuration and operational variables are crucial for the maximum yields of the process. Downstream processing. Cell disruption is a critical step to recover intracellular compounds and it affects recovery yield and carotenoids properties [ , ].
Carotenoid produced by yeasts as Rhodotorula spp. The developing of recovery techniques of carotenoids has been an issue of study. Some techniques used for carotenoid extraction are shown in Table 3. These techniques used for cell disruption allow obtain the carotenoid formed into the cells. Actually, the downstream processing is the major challenge to recover intracellular pigments; many investigations have been performed to concerning the recovery of products; an ideal recovery process must be in non-time-consuming, low cost and high yields.
The development of biotechnological processes to produce carotenoid has recently increased because it is a reliable method to obtain carotenoids. Several patents related to microbial production of carotenoids have been registered worldwide Table 4. A strategy to reduce the costs of production is the obtaining of hyper-producer strains. Efficient techniques have been described to obtaining of mutant strains [ ]. A simple technique is the random and selection mutagenesis through the color.
For this techniques have been used chemical Ethyl methane sulfonate, 1-methylnitroguanidine or antymicine A and physical methods UV light, gamma radiation. Metabolic engineering is the improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology [ ].
Nowadays, the engineering in non-carotenogenic microorganisms for carotenoid production is a very useful tool.
Metabolic engineering of microorganisms to produce higher levels of important molecules is a great possibility to improve public health through biotechnology [ ]. The selection of appropriate microorganism is the first step in biotechnological processes, which could be undergone to mutagenesis to improve the strains and subsequently the production of metabolites [ ].
Many authors have reported the application of metabolic engineering in yeasts, such as Sacharomyces cerevisiae [ — ] and Candida utilis [ , ]. These yeasts are very useful in food industries, they are considered generally recognized as safe organisms by FDA US [ ].
Bacteria such as Escherichia coli [ ] have been described as microorganisms engineered to produce carotenoid, however S. Although naturally the yeast S. Being a characterized its genetic system, physiology and regulatory networks [ ], this yeast is appropriate to be engineered to produce carotenoids. Verwaal et al. Ukibe et al. On the other hand, Pichia pastoris is other non-carotenogenic yeast that has also been studied to production of carotenoids, and it is able to grow in organic materials.
Araya-Garay et al. Carotenoids are important molecules that improve foods quality due to their high nutritional value. For these reasons, carotenoid application as colorants has increased the interest of industries and scientists to develop low-cost process for carotenoid production. Biotechnological production mainly provides economics advantages over synthetic or extracted plant carotenoids.
Yeast carotenoids have advantages such a high growth rate, decreasing the production time at industrial scale. On the other hand the use of low-cost substrates as agro-industrial wastes diminish the processes costs, and giving an alternative to the use of these wastes contributing to reduce environmental contamination. More research focused on the use of agro-industrial wastes, high carotenoids-producing yeast through metabolic engineering strategies, bioreactor design and control strategies for microbial carotenoids production are necessary to cover the worldwide demand by poultry, cosmetic, food, feed, beverages and others industries.
This review aims to show an overview on the biotechnological processes for carotenoid production, and the possibility to produce them at low costs, high yields and to reduce processing time. The knowledge about these processes is critical to improve and create new technologies in the production of microbial carotenoids.
Food Chem. CAS Google Scholar. Phytochemistry of fruit and vegetables. Oxford: Clarendon Press. Google Scholar. A Fiecher. Volume Edited by: Springer-Verlage. Heidelberg: Adv Biochl Eng Biotech. Prog Lipid Res. Technological and nutritional applications. Borowitzka MA: Micro-algae as sources of fine chemicals. Microbiol Sci. Appl Microbiol Biotechnol. Carotenoids, Volume 1a.
J Biosci Bioeng. J Biosc Bioeng. J Gen Microbiol. Biotech Lett. Strain ORS Xanthophylls contain oxygen atoms and are yellow pigments commonly found in nature [ 37 ].
Its both ionone rings carry hydroxyl groups [ 39 ]. Lutein is a substance that gives color to chicken fat, egg yolk, and chicken feathers [ 40 ]. That yellow-colored lutein found in plants is an organic colorant on leaves of green vegetables, such as spinach and black pepper. It is generally found in covalent interactions within fatty acids [ 20 ].
The chemical formulas of lutein and zeaxanthin are the same; in other words, they are isomers, but they are not stereoisomers. The difference between the two is the position of a double bond in the end ring [ 17 , 39 ].
Zeaxanthin is one of the most common carotenoid alcohols found in the nature. It is a pigment that gives its color to maize, saffron, and many other plants. When the zeaxanthin breaks down, picrocrocin, which is responsible for the taste and aroma of the saffron, forms [ 41 ].
Astaxanthin is a keto-carotenoid and is a zeaxanthin metabolite, containing both hydroxyl and ketone functional groups [ 17 , 43 ]. Fucoxantin, found in the chloroplasts of algae and mosses, gives them their brown or olive green color [ 45 ].
The interest in carotenoids found in plants over the last years is not only due to their A provitamin activity but also due to their reduction of oxidative stress in the organism by capturing oxygen radicals, that is, their antioxidant effects [ 46 ]. Free oxygen radicals play an important role in the stress-related tissue damage and pathogenesis of inflammation. The imbalance between protective and damaging mechanisms results in acute inflammation accompanied by neutrophil infiltration [ 47 , 48 ].
Superoxide radicals formed by neutrophils react with lipids and cause lipid peroxidation [ 49 — 51 ]. Carotenoids can inhibit active radicals by transferring electrons, giving hydrogen atoms to radicals or attaching to radicals [ 16 ]. The number of conjugate double bonds in their structure is closely related to the superoxide inhibitory effect of carotenoids [ 52 , 53 ].
Carotenoids could remove singlet oxygen and peroxyl radicals from reaction medium and also prevent their formation [ 53 — 55 ].
It is stated that carotenoids can inhibit cell renewal and transformation and regulate gene expression that plays a role in the formation of certain types of cancer. On the other hand, in some studies it has been shown that carotenoids could stimulate cancer in some cases. The activity of carotenoids as antioxidants also depends on their interaction with other antioxidants, such as vitamins E and C [ 58 ]. In addition, some carotenoids and their metabolites activate the nuclear factor-erythroid 2-related factor-2 Nrf2 transcription factor, which triggers antioxidant gene expression in certain cells and tissues [ 58 , 59 ].
Thus, a number of chronic diseases characterized by oxidative stress, inflammation, and impaired mitochondrial function have been reported to reduce Nrf2 expression in some animal models.
Elevation of Nrf2 has been shown to be effective in the prevention and treatment of many chronic inflammatory diseases, including various cardiovascular, renal, or pulmonary diseases; toxic liver damage; metabolic syndrome; sepsis; autoimmune disorders; inflammatory bowel disease; and HIV infection [ 58 ]. Lycopene, synthesized by many plants and microorganisms, is an antioxidant that cannot be synthesized by animals and humans [ 67 ].
Conjugated dienes are active in creating antioxidant activity [ 46 ], and lycopene is reported to have a higher antioxidant capacity than other carotenoids, and in particular among carotenoids, lycopene inhibits the risk of prostate cancer [ 52 , 68 ]. The use of lycopene can reduce the risk of cardiovascular disease, diabetes, osteoporosis, and prostate, esophagus, colorectal, and mouth cancer risk.
Recent studies indicate that lycopene intake has protective functions against cardiovascular diseases by lowering high-density lipoprotein HDL -associated inflammation [ 69 ].
It has also been suggested that lycopene bestow protection against amyotrophic lateral sclerosis ALS impairment in humans [ 67 ]. The phytoene and phytofluene found in the diet accumulate in the human skin [ 72 ]. The dehydration of these carotenoids has a protective effect on the skin due to its UV absorber, antioxidant, and anti-inflammatory properties [ 73 ]. Zeaxanthin is one of two carotenoids found in the retina. Zeaxanthin mostly found at the center of the macula and lutein mostly found at the peripheral retina [ 39 , 74 ].
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