Vitamins in plants: occurrence, biosynthesis and antioxidant function

Review

Vitamins in plants: occurrence, biosynthesis and antioxidant function M. Amparo Asensi-Fabado and Sergi Munne´-Bosch Departament de Biologia Vegetal, Universitat de Barcelona, Facultat de Biologia, Avinguda Diagonal 645, E-08028 Barcelona, Spain

Plant-derived vitamins are of great interest because of their impact on human health. They are essential for metabolism because of their redox chemistry and role as enzymatic cofactors, not only in animals but also in plants. Several vitamins have strong antioxidant potential, including both water-soluble (vitamins B and C) and lipid-soluble (vitamins A, E and K) compounds. Here, we review recent advances in the understanding of antioxidant roles of vitamins and present an overview of their occurrence within the plant kingdom, different organs and subcellular location; their major biosynthetic pathways, including common precursors and competitive pathways; and their antioxidant function. In particular, we discuss novel evidence for, as well as evidence against, a role of B vitamins as important antioxidants. The importance of vitamins Vitamins are compounds that cannot be synthesized by humans and thus need to be taken up in the diet. They have a complex biochemistry and play an essential role in human nutrition and health. Vitamin deficiencies cause diseases that can be severe and even lethal in some cases. For instance, vitamin A deficiency is a major health problem in low-resource countries, putting an estimated 125– 130 million children at increased risk of morbidity and mortality from infectious diseases [1]. Well-known human vitamin-related disorders include, among others, blindness (vitamin A), beriberi (vitamin B1), pellagra (vitamin B3), anemia (vitamin B6), scurvy (vitamin C) and rickets (vitamin D). In much of the developed world, such deficiencies are, however, rare due to an adequate supply of food and/or the addition of vitamins to common foods, often called fortification. For instance, the deficiency of folic acid (vitamin B9) that generally occurs during pregnancy is avoided through supplementation, therefore preventing neural tube defects in the fetus [2]. Alternatively, vitamins in plant-derived foods can also be increased either through an optimization of growth conditions, conventional plant breeding or through use of transgenic techniques, a process known as biofortification [3]. Plants contain a wide range of vitamins that are essential not only for human metabolism but also for plants, because of their redox chemistry and role as cofactors, and some of them also have strong antioxidant potential (Figure 1, Table 1, Box 1). The antioxidant vitamins that have been the focus of most attention in plants are carotenoids (pro-vitamin A), ascorbate (vitamin C) and tocochromanols (vitamin E, including both tocopherols and tocotrienols) (reviewed in [4–11]). However, recent Corresponding author: Munne´-Bosch, S. ([email protected]).

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evidence indicates that vitamin B compounds could also play a significant role as antioxidants in plants. Thiamine (vitamin B1) has been shown to alleviate the effects of several environmental stresses on Arabidopsis (Arabidopsis thaliana), presumably by protecting the plant from oxidative damage [12]. The antioxidant role of thiamine can be indirect, by providing NADH and NADPH to the antioxidant network, or direct, by acting as an antioxidant. Another important finding is increased sensitivity to photooxidative stress in vitamin B6-deficient Arabidopsis plants [13]. Pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivates are collectively known as vitamin B6. Singlet oxygen levels increase in the pdx1 mutant, which is deficient in de novo vitamin B6 biosynthesis. Furthermore, the pdx1 mutation enhances the photosensitivity of the vte1 npq1 Arabidopsis double mutant, which indicates interplay between vitamin B6, tocopherols and carotenoids in chloroplasts [13]. Other compounds with potential antioxidant activity within the plant cell include folates (vitamin B9) and phylloquinone (vitamin K1). This review focuses on the occurrence, biosynthesis and antioxidant function of vitamins in plants, including classical groups (vitamins A, C and E) and other compounds (vitamins B and K). We emphasize the common points of their biosynthetic pathways and

Glossary Antioxidant: a molecule with a low reduction potential that can donate electrons or hydrogen atoms thereby preventing in low concentrations (i.e. in the magnitude of mg/g) the oxidation of other molecules. Antioxidant vitamin: a vitamin (see below) with antioxidant properties. Cofactor: a compound that needs to be present in addition to an enzyme for a certain reaction to occur. Oxidative damage: injury to the cell caused by oxidative stress (see below). Oxidative stress: an imbalance between ROS (see below) and antioxidants. Pro-vitamin: a substance that is converted into a vitamin in animal tissues. ROS: reactive oxygen species produced from aerobic metabolism, including superoxide anions (O2–), hydrogen peroxide (H2O2), hydroxyl radicals (OH–) and singlet oxygen (1O2), which are important components of the intracellular signalling network but which, when allowed to accumulate at high concentrations, can damage cellular lipids, proteins and DNA. ROS scavenging: chemical reaction of an antioxidant on any ROS. Singlet oxygen quenching: physical reaction that leads to the de-excitation (removal) of several molecules of singlet oxygen while the antioxidant is consumed in low amounts. TEAC assay: Trolox equivalent antioxidant capacity assay used to estimate the antioxidant capacity of molecules. An antioxidant is added to a free radical generating system and the inhibition of the free radical action is measured. This inhibition is related to the antioxidant capacity of the sample in relation to that of the water-soluble vitamin E analog Trolox1. Tocochromanol: tocopherols and tocotrienols are collectively known as tocochromanols. They form the vitamin E group of compounds. Vitamin: any of various fat-soluble or water-soluble organic substances essential in minute amounts for normal growth and activity that cannot be synthesized in the organism for which they are ‘vital’ and obtained naturally from plant and animal foods.

1360-1385/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2010.07.003 Trends in Plant Science, October 2010, Vol. 15, No. 10

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Figure 1. Chemical structures of vitamins found in plants.

discuss which vitamins can be considered antioxidants in plants in vivo. Occurrence Vitamins B, C and K1 are ubiquitous in plants [14–16]. By contrast, although tocopherols and b-carotene, the main vitamin A biosynthetic precursor, are present in all photo-

synthetic organisms, other forms of pro-vitamin A (a-carotene and b-cryptoxanthin) and tocotrienols are not uniformly distributed within the plant kingdom [10,17]. Table 2 summarizes the occurrence of vitamins in plant organs. Fruit is the main source of vitamin C: for example, guava (Psidium guajava) fruit contain up to 2.28 mg/g of ascorbate. Leaves are also a good source of vitamin C: the 583

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Table 1. Antioxidant activity of plant-derived vitamins Vitamin Pro-vitamin A

B1 B2 B3 B5 B6

B7 B9

C E K1

Compound(s) a-Carotene b-Carotene b-Cryptoxanthin Thiamine Riboflavin Niacin Pantothenic acid Pyridoxal Pyridoxine Pyridoxamine Biotin Folic acid Dihydrofolate Tetrahydrofolate 5-Methyltetrahydrofolate Ascorbic acid Tocopherols Tocotrienols Phylloquinone

Solubility Lipid-soluble Lipid-soluble Lipid-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Water-soluble Lipid-soluble Lipid-soluble Lipid-soluble

TEAC value 1.300.04 1.900.10 2.000.02 0.320.01 Unknown Unknown Unknown 0.030.00 0.030.00 0.030.00 Unknown 0.060.01 0.980.01 0.730.04 0.770.04 0.990.05 0.970.03 0.980.04 Unknown

Antioxidant capacity O2 quencher 1 O2 quencher 1 O2 quencher O2–/OH– scavenger None described None described None described O2–/1O2 scavenger O2–/1O2 scavenger O2–/1O2 scavenger None described O2–/1O2 scavenger O2–/1O2 scavenger O2–/1O2 scavenger O2–/1O2 scavenger H2O2 /OH– /1O2 scavenger 1 O2 quencher 1 O2 quencher LOO–/LO–/HO–scavenger

Refs [81–83]

1

[84,85]

[58,68]

[74,86]

[55,87] [88,89] [80]

TEAC, Trolox equivalent antioxidant capacity.

ascorbate concentration in cabbage (Brassica oleracea) leaves can be up to 0.58 mg/g (Danish Food Composition Databank: http://www.foodcomp.dk/v7/fcdb_details.asp? FoodId=0674). Dry seeds and dormant buds have little or no ascorbate, but they retain some dehydroascorbate [15]. b-Carotene mostly accumulates in leaves, with a concentration in the range of 10–60 mg/g. a-Carotene and b-cryptoxanthin are less abundant and are mainly found in fruits such as pumpkins (Cucurbita spp.) (USDA National Nutrient Database: http://www.ars.usda.gov/ba/ bhnrc/ndl). Carrot (Daucus carota) is a remarkable root vegetable that contains higher concentrations of a- and b-carotene than those found in leaf vegetables (Danish Food Composition Databank: http://www.foodcomp.dk/v7/ fcdb_foodcomplist.asp?CompId=0016). b-Cryptoxanthin is particularly found in fruits, but also in seeds and flowers [17–20]. Plant tissues vary enormously in their vitamin E content and composition. Photosynthetic tissues generally contain low levels of vitamin E (<50 mg/g) compared with seeds and particularly oil seeds, which contain 10–20 times this level. High levels of tocotrienols are present in the fruits and seeds of some species: the oils that are obtained from these species have renowned health properties, such

as palm oil (Elaeis guineensis) [21]. Most green vegetables contain high concentrations of phylloquinone (vitamin K1), although this compound can also be found at lower concentrations in seeds [e.g. wheat (Triticum aestivum), peas (Pisum sativum)], fruits [e.g. strawberries (Fragariaananassa), cucumber (Cucumis sativus)] and tubers [e.g. potato (Solanum tuberosum)]. Among root vegetables, only carrots contain significant amounts of phylloquinone [22]. Like phylloquinone, the highest concentrations of B group vitamins in plant tissues are often found in a range of several mg/g, with the notable exception of biotin, which is found at concentrations in the range of ng/g fresh wt. B group vitamins mainly accumulate in seeds, such as in those of alfalfa (Medicago sativa, Danish Food Composition Databank: http://www.foodcomp.dk/v7/fcdb_details.asp? FoodId=0868). The bran of certain seeds contains remarkable concentrations of vitamins B1 and B3. For example, rice bran (Oryza sativa) contains 27.5 mg/g vitamin B1 and 340 mg/g vitamin B3, whereas polished rice has only 0.7 mg/ g vitamin B1 and 14 mg/g vitamin B3. Wheat bran also contains a fivefold higher concentration of vitamin B3 (296 mg/g) compared with that found in wheat germ. By contrast, vitamins B2, B3 and B9 can be found at similar

Table 2. Occurrence of vitamins in plant organs Vitamin Pro-vitamin A

B1 B2 B3 B5 B6 B7 B9 C E K1

Compound(s) a-Carotene b-Carotene b-Cryptoxanthin Thiamine Riboflavin Niacin Pantothenic acid Pyridoxal Biotin Folates Ascorbate

Leaves + ++ + + + + ++ + + ++ ++

Flowers – + + + + + + + + + +

Fruits ++ + ++ + + + + + ++ + ++

Seeds + + + ++ ++ ++ ++ ++ ++ ++ +

Roots + + – + + + + + + + +

Tubers – + + + + + + + + + +

Bulbs – + – + + + + + + + +

Tocopherols Tocotrienols Phylloquinone

+ – ++

+ – +

+ + +

++ ++ +

+ – +

+ – +

+ – +

Key: +, vitamin present; ++ vitamin present in high amounts relative to other organs; , vitamin absent or yet not found.

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Box 1. Calciferol, cobalamin and rutin Calciferol Vitamin D is a group of lipid-soluble compounds, the two major forms of which are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Ergocalciferol and cholecalciferol are obtained from ergosterol and cholesterol after sun exposure following two hydroxylation reactions in the body [90]. Whereas mammalian and fungal cells generally contain one major sterol, cholesterol and ergosterol, respectively, plants have a characteristically complex sterol mixture, including sitosterol, stigmasterol and 24-methylcholesterol. For example, 24-methylcholesterol leads to brassinosteroid biosynthesis [91]. Cholesterol is only a minor sterol in most plant species, but it is present at relatively high levels (approximately 15% to 20% of total sterols) in Solanaceous plants such as potato (Solanum tuberosum) and tobacco (Nicotiana tabacum). It is a crucial precursor of glycoalkaloids, steroidal toxic secondary metabolites produced as defense compounds upon wounding that are toxic for humans [92]. Given that the human body also has the capacity to synthesize cholesterol, plants cannot be considered a source of pro-vitamin D (Figure I).

Cobalamin Cobalamin, also called vitamin B12, is a water-soluble vitamin that it is unique in being exclusively synthesized by prokaryotes. Humans primarily obtain it from meat, thus vegetarians can suffer a deficiency, particularly if they do not consume eggs. Although it has outstanding antioxidant properties in vitro [93], it is not found in plants and only some algae require it. In algae, it is obtained from symbiotic associations with bacteria, as it is in herbivores [94]. Rutin Rutin, also called rutoside or quercetin-3-rutinoside, is a glycoside flavonoid with outstanding antioxidant properties. It is superior to vitamin E in the TEAC assay and accumulates in several plant species [95]. Although sometimes erroneously called vitamin P, rutin and other flavonoids are not vitamins because, despite their beneficial effects, they have not been shown to be essential for human health.

[(Figure_I)TD$G]

Figure I. Chemical structures of cholecalciferol (vitamin D3), cobalamin (vitamin B12) and rutin.

concentrations in leaves and seeds. In general, biotin is the least abundant B vitamin in plants, although some vegetables contain relatively high concentrations [e.g. kale (Brassica oleracea), wheat bran, walnuts (Juglans regia), banana (Musa) and carrot] (Danish Food Composition Databank: http://www.foodcomp.dk/v7/fcdb_search.asp). Ascorbate is present in most, if not all, cell compartments in higher plants. For example, as much as 30% of the total leaf ascorbate content can accumulate in chloroplasts [23]. Substantial amounts of ascorbate are also found in the apoplast [24], cytosol and vacuoles [25]. Vitamin E is found in chloroplasts and other plastids, and in cytoplasmic lipid bodies in seeds [26]. Similarly, lipid-soluble phylloquinone is not only restricted to chloroplasts but it has also been

found in plasma membrane preparations [27]. Vitamin B compounds are found in the cytosol, plastids and mitochondria [14]. Biosynthesis The elucidation of vitamin biosynthesis in plants has been helped enormously by our knowledge of bacterial pathways, except in the case of vitamin C, which is synthesized exclusively by eukaryotes. The subcellular compartmentalization of eukaryotic cells makes the distribution and cellular trafficking of numerous metabolites more complex. This is particularly true of plants, which together with eukaryotic algae are unique in containing plastids. In plants, the biosynthesis of some vitamins is restricted to 585

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Figure 2. Cross-points on the biosynthetic pathways of vitamins in plants. The subcellular location of the reactions in the white boxes is unknown. Shared precursors of several vitamin biosynthetic pathways are depicted in colored boxes. Enzyme activities that divert the shared precursors towards a specific vitamin biosynthetic route (competitive pathways) are numbered: (1) 1-deoxy-D-xylulose-5-phosphate synthase; (2) 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; (3) 5-phosphoribosyl-1pyrophosphate synthase; (4) 3,4-dihydroxy-2-butanone 4-phosphate synthase; (5) quinolinate synthase; (6) pyridoxal 50 -phosphate synthase; (7) phosphomannose isomerase; (8) chorismate mutase; (9) isochorismate synthase; (10) 4-amino-4-deoxychorismate synthase; (11) phytoene synthase; (12) homogentisate geranylgeranyl transferase; (13) geranylgeranyl reductase; (14) homogentisate phytyl transferase; (15) 1,4-dihydroxy-2-naphtoate phytyl transferase; (16) lycopene e-cyclase; (17) lycopene b-cyclase; (18) b-ring hydroxylase; (19) 5-aminoimidazole ribonucleotide carboxylase; (20) THIC protein; (21) GTP cyclohydrolase II; (22) GTP cyclohydrolase I; (23) thiazole synthase (THI1); (24) glycine amide ribonucleotide synthase; (25) hydroxyphenylpyruvate (HPP) dioxygenase. For ease of visualization, the names of the vitamins are displayed next to the corresponding metabolic compounds. Lipid-soluble vitamins are depicted in red, water-soluble vitamins are depicted in blue. The activity of vitamins as cofactors in some enzymatic reactions is indicated in blue. Abbreviations: AIR, 5-aminoimidazole ribonucleotide; HET-P, 4-methyl-5-b-hydroxyethyl thiazole phosphate; HMP-PP, 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate; HPP, hydroxyphenylpyruvate; IMP, inosine monophosphate; PPRP, 5-phosphoribosyl-1pyrophosphate; S-AdoMet, S-adenosylmethionine.

one compartment. For example, carotenoids (pro-vitamin A), vitamins E and K1 and water-soluble riboflavin are produced in the plastids [28–30]. However, some enzymes of phylloquinone biosynthesis have recently been found in peroxisomes [31] and riboflavin is further converted to flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in the cytosol, plastids or mitochondria [32]. Interestingly, the biosynthesis of the remaining watersoluble vitamins is split between different compartments, including the mitochondria [14] (Figure 2). The upstream precursors for most vitamins come from carbohydrate metabolism, which regulates the pools of hexoses, pentoses and trioses in the plastids and the cytosol. The pentose and triose pool in the plastids provides: (a) erythrose-P and phosphoenolpyruvate for the synthesis of chorismate, the common intermediary in the biosynthesis of tocochromanols, phylloquinone and folates [33–35]; (b) 586

glyceraldehyde 3-P and pyruvate (from phosphoenolpyruvate), which are required for the synthesis of geranylgeranyl-PP, a key shared precursor of lipid-soluble vitamins [28,33]; (c) ribulose 5-P for the synthesis of riboflavin [36]; (d) ribose 5-P, which is related to thiamine, riboflavin and folates through purine biosynthesis [14]; and (e) dihydroxyacetone-P for niacin [14]. In the cytosol, fructose 6-P feeds the main de novo route to ascorbate biosynthesis: the Lgalactose pathway [37]. The interconvertible trioses- and pentoses-P glyceraldehyde 3-P/dihydroxyacetone-P and ribose 5-P/ribulose 5-P, respectively, are the precursors of pyridoxal 50 -phosphate [38]. The compounds in the carbohydrate pool can be interconverted by means of a range of enzymatic activities, which keep the cytosolic and plastid-localized pools in equilibrium [39]. Therefore, the carbohydrate pools and the metabolic pathways that feed and drain them (the Calvin cycle, the pentose phosphate

Review pathway and glycolysis) might constitute the top control point for determining the fluxes of precursors towards the different branches that lead to the aforementioned vitamins. Downstream of carbohydrate metabolism, several branching points are readily identified in plastids. These include: chorismate, which is the common precursor of folates through the synthesis of p-aminobenzoate [35]; phylloquinone through its conversion to isochorismate [34]; and tocochromanols upon synthesis of homogentisate [33]. Geranylgeranyl-PP appears to be another key branch point from which the flux is diverted towards either of the lipid-soluble vitamins. This metabolite can follow the route leading to phytoene, which is used in carotenoid biosynthesis [17,28]. Otherwise, complex branching starts from this point: geranylgeranyl-PP leads to tocotrienol biosynthesis upon condensation with homogentisate, whereas conversion to phytyl pyrophosphate (phytyl-PP) and posterior condensation with homogentisate leads to tocopherols [10,11,33]. Another shared precursor of vitamins in the plastids is 5-aminoimidazole ribonucleotide (AIR). AIR is an intermediary in purine biosynthesis that can either give rise to the pyrimidine moiety of thiamine (HMP-PP) in the plastids or follow the purine biosynthetic route that leads to guanosine-phosphate in the cytosol, followed by conversion to GTP, which in turn is a common precursor of riboflavin and folates [14]. Interestingly, the amino acid glycine is a common precursor of AIR and the thiazole moiety of thiamine [14]. However, the remaining watersoluble vitamins (B5, B6, B8 and C) have no shared precursors downstream of the aforementioned carbohydrate pools. Nevertheless, vitamins B1, B6, B9 and C are required as cofactors in the metabolism of other vitamins. Hence, thiamine pyrophosphate (PP) participates in the route that leads to geranylgeranyl-PP and is therefore involved in the biosynthesis of lipid-soluble vitamins [40]. Pyridoxal 50 -phosphate is also a cofactor in the biotin biosynthetic pathway [14,41]. Methyltetrahydrofolate is involved in the biosynthesis of several vitamins, such as pantothenate, phylloquinone, thiamine and riboflavin, through its participation in the purine biosynthetic pathway [14]. Similarly, S-adenosylmethionine, which is closely linked to folates, is required for tocopherol and tocotrienol biosynthesis [14]. Ascorbate is a cofactor of hydroxyphenylpyruvate dioxygenase, a key enzyme leading to homogentisate and therefore to tocochromanol biosynthesis [42]. Finally, it is noteworthy that amino acid metabolism plays a role in vitamin biosynthesis because amino acids are precursors of tocochromanols, thiamine, niacin, pantothenate, pyridoxal 50 -phosphate and biotin. Glutamine and glutamate participate in a range of reactions within the specific and shared biosynthetic pathways of several vitamins, such as pyridoxal 50 -phosphate, folates and the purine pathway that connects thiamine, riboflavin and folates. The transgenic expression of vitamin biosynthetic genes has been investigated for overproduction of these compounds in plants and successfully achieved with vitamins A, C, E, B6 and B9 [33,37,43–45]. However, to date, relatively few attempts have been made to manipulate different vitamins simultaneously in plants, even though multigene engineering offers an excellent opportunity to

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do this [46]. Furthermore, existing approaches have also demonstrated the unpredictability associated with engineering plant metabolic pathways. By transformation with antisense constructs encoding zeaxanthin epoxidase, in which zeaxanthin conversion to violaxanthin is inhibited, not only zeaxanthin levels increased but also tocopherol levels were elevated in potato tubers [47]. Other positive examples include the high tocopherol levels in the high bcarotene varieties of tomato generated through the constitutive overexpression of a bacterial phytoene desaturase [45]. However, in other cases, an increase in the level of one vitamin leads to a decrease in the level of another vitamin. For instance, overexpression of VTE1 in Arabidopsis, which encodes for tocopherol cyclase, led to several-fold increases in vitamin E (in the form of g-tocopherol) but vitamin C levels were reduced by 60% [48]. Furthermore, plants often show numerous pleiotropic effects such as cosuppression or sense suppression resulting in loss of plant vigor. One of the most striking examples is the severe dwarfism brought about by manipulating transgenic tomato (Solanum lycopersicum) plants to produce increased levels of carotenoids owing to a redirection of metabolites away from the gibberellin pathway [49]. Finally, it should be borne in mind that some vitamins, such as vitamins C and B6, participate in hormone biosynthesis and signalling [50–52], which in turn influence vitamin synthesis in plants [53,54], thus reflecting complex interactions between vitamins and hormones. Increasing levels of vitamins in plants is not only important for biofortification but also because they display important antioxidant functions, which might improve redox balance and therefore benefit crop growth and resistance to both biotic and abiotic stresses. Antioxidant function The most abundant and ubiquitous cellular antioxidant is ascorbate (vitamin C). Ascorbate plays a prominent role in the antioxidant defense network of plants because of its excellent ability to scavenge reactive oxygen species (ROS). It acts in coordination with glutathione and enzymatic antioxidants in chloroplasts, mitochondria, peroxisomes and cytosol in the ascorbate–glutathione cycle to control the amount of hydrogen peroxide formed within the cell. Thus, it plays a major role in cellular signalling and in preventing oxidative damage to macromolecules (reviewed in [55,56]). Ascorbate is also found in significant amounts in the apoplast, where it acts in concert with other antioxidants to control the cellular redox state and orchestrates multiple stress responses within the cell [56,57]. Carotenoids and tocopherols control ROS accumulation in plastids, thereby playing a major role in controlling singlet oxygen levels within thylakoid membranes. Carotenoids counteract the chlorophyll-photosensitized formation of singlet oxygen by intercepting (de-exciting or quenching) (i) chlorophyll triplet states and (ii) singlet oxygen once formed (reviewed in [58]). a-Tocopherol affords protection to membranes mainly by quenching singlet oxygen and reacting with lipid peroxy radicals and has been shown to reduce the extent of lipid peroxidation in leaves and seeds [59,60]. Recent evidence [12,13] suggests that vitamins other than ascorbate, carotenoids and tocopherols also play a 587

Review significant role as antioxidants in plant cells. The clearest evidence has been obtained for vitamin B6 [13,61,62]. Although the Trolox equivalent antioxidant capacity (TEAC) assay (see Glossary) gives low antioxidant values for vitamin B6, it is a potent antioxidant that is equivalent to vitamins C and E and is particularly active in scavenging singlet oxygen and superoxide anions [63–65]. Pyridoxal 50 -phosphate, in particular, is well established as a cofactor for enzymes involved in amino acid, lipid and carbohydrate metabolism [66,67], and the free forms of the vitamin B6 group are among the most potent antioxidants [68]. Vitamin B6, however, gives low TEAC values because this is a measure of the protective action of antioxidant compounds against free radicals and these compounds are efficient in singlet oxygen quenching/ scavenging but not in trapping free radicals [58]. PDX1 gene expression is enhanced by high light treatments [61] and pdx1 mutants are hypersensitive to Rose Bengal, a photosensitizer that can generate singlet oxygen upon illumination [62], an effect that has not been observed with superoxide anions or hydrogen peroxide [13]. Furthermore, the maximum efficiency of photosystem II (PSII) photochemistry (F v/F m) decreases concomitantly with a reduction in the D1 protein in pdx1 mutants that are exposed to high light [61]. A fraction of the vitamin B6 pool must be located close to PSII for efficient quenching of singlet oxygen at its site of production and for prevention of D [(Figure_3)TD$IG] 1 oxidation, which is supported by the recent localization

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of vitamin B6 in chloroplasts of tobacco [13]. Furthermore, a triple Arabidopsis mutant (vte1 npq1 pdx1) that is deficient in tocopherols, zeaxanthin and vitamin B6 exhibited increased sensitivity to high light, with severe pigment loss and enhanced lipid peroxidation [13]. This study also showed that pdx1 mutants exposed to high light have lower tocopherol levels than the wild type, as well as higher singlet oxygen production and lipid peroxidation. This suggests that more tocopherols are consumed in vitamin B6-deficient plants. Similarly, the vtc1 mutant of Arabidopsis, which has only 40% of wild-type levels of ascorbate in the chloroplasts, exhibits enhanced lipid peroxidation and a severe depletion of a-tocopherol and b-carotene levels in response to water deficit [69]. Furthermore, ascorbate is a cofactor needed for violaxanthin de-epoxidase activity, and therefore its deficiency in vtc1 mutants limits dissipation of excess excitation energy in chloroplasts, which also increases sensitivity of plants to photooxidative stress [70]. Taken together, the results obtained to date indicate that carotenoids and vitamins B6, C and E act in concert to control singlet oxygen levels in chloroplasts, thereby protecting plants against photooxidative stress (Figure 3). Although these vitamins might apparently show an overlapping, redundant antioxidant role, their function in plants is remarkably complementary. Vitamin C is ubiquitously present and it is an excellent cellular redox state sensor that controls the most readily diffusible ROS,

Figure 3. Model of the antioxidant function of vitamins in chloroplasts. Vitamins participate either directly or indirectly in photosynthetic electron transport: phylloquinone (vitamin K1) as an integral part of the redox reactions of PSI and thiamine (vitamin B1) as a precursor of NADP(H). Carotenoids (pro-vitamin A), ascorbate (vitamin C), tocopherols (vitamin E) and pyridoxal and derivatives (vitamin B6) play a major role in the elimination of ROS in chloroplasts, particularly in the removal of singlet oxygen produced in PSII and hydrogen peroxide produced in PSI (indicated by red arrows). Vitamins B1 and C also participate in antioxidant (AOX) recycling (blue arrows). Abbreviations: AOX, antioxidant; Cyt, Cytochrome; Fd, Ferredoxin; FNR, Ferredoxin–NADP reductase; PC, plastocyanin; PQ, plastoquinone; PSI, photosystem I; PSII, photosystem II; ROS, reactive oxygen species.

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Review hydrogen peroxide and helps the photo- and antioxidant protection machinery by acting as a cofactor of violaxanthin de-epoxidase and presumably by recycling oxidized tocopherol. Carotenoids also appear to be particularly effective in preventing the accumulation of singlet oxygen, a function that is also carried out by tocopherol. Different chemical properties of carotenoids and vitamin E optimize singlet oxygen quenching by acting at different locations in chloroplasts, not only at the site of production in PSII but also inside the lipid matrix of thylakoids. Furthermore, vitamin E seems to play an essential role in preventing the propagation of lipid peroxidation. Vitamin B6 also appears to be effective in scavenging singlet oxygen but its presence outside the chloroplasts and its role as cofactor clearly shows that the role of this vitamin is more complex, similarly to what occurs with vitamin C. It has been shown that SOS4, a pyridoxal kinase that generates pyridoxal 50 -phosphate, is required for salt tolerance in A. thaliana, most probably associated with an effect on Na+ and K+ homeostasis [71]. PDX1 was found to be mainly associated with the membrane system of cells, including the plasma membrane, cytosol, nuclear envelope and chloroplast outer membranes [62]. As what happens with vitamin C, roots of pdx1 mutants are impaired both in cell division and in cell elongation and both vtc1 and pdx1 mutants have similar stunted root phenotypes. However, supplementation with vitamin C failed to recover the growth of pdx1 roots [62]. It appears, however, that this effect is related to a defect in auxin biosynthesis rather than to a direct antioxidant role, because vitamin B6 is required for the biosynthesis of tryptophan, a precursor of auxin biosynthesis, which is indeed involved in root growth [52]. It appears, therefore, that pyridoxal 50 -phosphate, in particular, acts as an important cofactor and therefore controls essential processes of growth and development as well as plant responses to stress, whereas the free forms of the vitamin B6 group, which show antioxidant properties, might contribute to the antioxidant defense network of plants in vivo. Another vitamin that might be a potent antioxidant in vivo is thiamine (vitamin B1). In a recent study, paraquattreated Arabidopsis plants showed reduced amounts of protein carbonyls and dichlorofluorescein diacetate staining, which are two indicators of oxidative stress, when thiamine was applied [12]. This study also showed that the levels of thiamine, thiamine monophosphate and thiamine pyrophosphate increased in Arabidopsis seedlings that were exposed to high light, low temperatures, osmotic and salt stress or paraquat. These increases were associated with an increase in the transcript levels of some genes in the thiamine biosynthetic pathway by up to sixfold. Furthermore, at least part of this biosynthetic pathway was enhanced in high light-exposed apx1 mutants, which were deficient in ascorbate peroxidase and were therefore more sensitive to oxidative stress [12]. The cofactor form of thiamine, thiamine pyrophosphate, is by far the most abundant vitamin B1 found in plants. Hence, it has been suggested that thiamine plays an indirect role as an antioxidant in plants by providing NADH and NADPH to combat oxidative stress [12]. Although thiamine is a potent scavenger of superoxide anions and hydroxyl radicals [72],

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and could also protect membranes from lipid peroxidation [73], it is still unclear whether these protective effects are the result of a direct antioxidant effect. Folates (vitamin B9) are another vitamin group with outstanding antioxidant properties. Studies of the antioxidant activity of folic acid and its reduced forms, 7,8-dihydrofolate, 5,6,7,8-tetrahydrofolate and 5-methyltetrahydrofolate, reveal that although folic acid can be considered a poor antioxidant, the reduced forms present outstanding antioxidant properties in vitro that are comparable to vitamins C and E [74]. Furthermore, reduced forms of folic acid are efficient at scavenging peroxynitrite and in inhibiting lipid peroxidation [74–76]. However, reduced forms of folic acid are also active as cofactors in the transfer and use of one-carbon groups. They donate one-carbon group in the biosynthesis of purine, pyrimidine and DNA and play a key role in the regeneration of methionine and in the biosynthesis of vitamin B5 [77,78]. They are indirectly involved in the biosynthesis of S-adenosylmethionine and therefore of all isoprenoids (including carotenoids and vitamin E). Consequently, it is difficult to discern between the direct and indirect effects of their antioxidant function in vivo. Other vitamins are involved in the regulation of the cellular redox state and can therefore play a role in the plant antioxidant defense network. Riboflavin (vitamin B2) is primarily found as an integral component of the coenzymes FAD and FMN, and it participates in multiple redox reactions [30]. Niacin, also known as vitamin B3 or nicotinic acid, is converted to nicotinamide and then to NAD and NADP in vivo. Thus, it also plays a key role in cellular redox chemistry [32]. Finally, phylloquinone (vitamin K1) is an essential component of photosynthetic electron transport, in which it constitutes the second step in the PSI redox chain [79]. Phylloquinone has potent antioxidant activity in solvents and isolated membranes and also acts in redox reactions at the plasma membrane of plants [80]. However, further research is needed to clarify the roles of vitamins B and K in the antioxidant defense network of plants. Concluding remarks and perspectives Vitamins are important regulators of cellular metabolism in plants, as cofactors in many enzymatic reactions but also as antioxidants. Studies on the distribution and occurrence of vitamins in plants clearly indicate that they are widely distributed in the plant kingdom, in several, if not all, organs and in most cellular compartments. Research has revealed several common crossing points, as well as the enzymes and pathways that are involved in their biosynthesis, which will help us to simultaneously manipulate contents of several vitamins in plant foods. However, the complexity of competitive pathways of vitamin biosynthesis in plants is still poorly understood and makes this goal extraordinarily challenging. Plastids appear to be one of the organelles that are most protected by antioxidant vitamins. This is not surprising because they sustain life through oxygenic photosynthesis and are therefore highly exposed to oxidative stress. The evidence indicates that in addition to classical antioxidants such as carotenoids and vitamins C and E, other vitamins form part of the antioxidant network of plants. Vitamins B1 589

Review and B6 appear to play an important role in plant resistance to oxidative stress. One of the major challenges in the near future will be to discriminate between the direct and indirect effects of these vitamins on plant stress tolerance. Vitamins B1 and B6 are excellent examples of how difficult it is to disentangle the cellular mechanisms by which these compounds confer plant responses to oxidative stress, either through an indirect effect as cofactors or as a direct effect as antioxidants. Other vitamins such as vitamin B9 and vitamin K1 also show outstanding antioxidant capacity in vitro. Therefore, they offer an additional excellent opportunity for future studies on the antioxidant function of vitamins in plants. Disclosure statement The authors declare no conflicting interests. Acknowledgments Research in the S.M-B. laboratory (Departament de Biologia Vegetal, Universitat de Barcelona) is supported by BFU2006-01127, BFU200907294, BFU2009-06045 and CSD2008-00040 grants from the Spanish Ministry of Science and Innovation, A/026613/09, A/019625/08 and A/ 016255/08 grants from the AECI (Agencia Espan˜ola de Cooperacio´n Internacional), and the prize ICREA (Institucio´ Catalana de Recerca i Estudis Avanc¸ats) Academia, which is funded by the Generalitat de Catalunya. We thank Jon Falk, Karin Schwarz and Maren Mu¨ller for their critical review of the manuscript.

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