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Research article Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature Simona Proietti a, 1 , Stefano Moscatello a, 2 , Franco Famiani b, 3 , Alberto Battistelli a, * a Istituto di Biologia Agroambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche (CNR), Viale Marconi 2, 05010 Porano (TR), Italy b Dipartimento di Scienze Agrarie e Ambientali, Universita ` degli Studi di Perugia –Borgo XX Giugno, 74, 06121 Perugia, Italy article info Article history: Received 23 December 2008 Accepted 19 March 2009 Available online 28 March 2009 Keywords: Acclimation Ascorbic acid Oxalic acid Low temperature Spinach abstract The effect of acclimation to 10 C on the leaf content of ascorbic and oxalic acids, was investigated in spinach (Spinacia oleracea L.). At 10 C the content of ascorbic acid in leaves increased and after 7 days it was about 41% higher than in plants remaining under a 25 C/20 C day/night temperature regime. In contrast, the content of oxalate, remained unchanged. Transfer to 10 C increased the ascorbic but not the oxalic acid content of the leaf intercellular washing fluid (IWF). Oxalate oxidase (OXO EC 1.2.3.4) activity was not detected in extracts of leaf blades. Therefore, oxalic acid degradation via OXO was not involved in the control of its content. Our results show that low temperature acclimation increases nutritional quality of spinach leaves via a physiological rise of ascorbic acid that does not feed-forward on the content of oxalic acid. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction Plant foods provide humans with most of their energetic and nutritional needs but can also be the major source of nutritionally negative compounds. Ascorbic and oxalic acids are two related plant metabolites with opposite nutritional value for humans. While ascorbic acid (vitamin C) intake with plant food is highly positive and can fulfil the daily intake need for humans [27], soluble and insoluble oxalate in the human and animal diet poses nutri- tional and health problems [37]. Soluble oxalic acid present in food chelates cations in the digestive trait and this decreases their intestinal absorption. High oxalic acid in the blood, that might depend on food intake, increases the risk of kidney stones [37], a large proportion of which are composed of calcium oxalate. Ascorbic acid present in food is thought to partially counteract this negative effect of oxalic acid [37]. Ascorbic acid is present in all plant species and tissues where it plays a crucial role in energy metabolism, growth, development, response to biotic and a-biotic stresses and flowering [3]. Ascorbic acid can be synthesised by multiple pathways, actively studied world wide, and its turnover can be very fast, although relatively little is known about the contribution of ascorbic acid degradation to the control of ascorbic acid content of plant tissues [7] particu- larly under environmental stress conditions. Oxalic acid is a widespread metabolite in the plant kingdom. It can be found as a soluble acid or insoluble salt, mainly as calcium oxalate. Considerable variations occur in the distribution of soluble and insoluble oxalate in plants, depending on the species, variety, age, tissues, [21,26] growth environment [33] and agricultural practices [40]. Several important roles for the physiology of the plant are now attributed to oxalic acid and to calcium oxalate. Oxalic acid is involved in the cellular pH stat [26], and it has been connected to nitrate reduction [1] and also involved in the oxidative reactions in the apoplast after pathogen invasion [19]. Insoluble calcium oxalate plays a role in osmoregulation [23,28] in defence against grazing animals, in the detoxification of heavy metals (and oxalic acid). It also affects light distribution in leaves [26,27] and it is the product of excess calcium sequestration in idioblast [9,22]. Oxalic acid in plant can be synthesised by different pathways [9], but the oxidative degradation of ascorbate that involves cleavage of the C 2 –C 3 bond of the L-ascorbic acid carbon chain, is now indicated as the most important one [22,23,27]. This is known to occur in the specialised cells idioblasts, where the breakdown of ascorbic acid produces the oxalic acid used for the formation of the calcium Abbreviations: AsA, ascorbic acid; DAsA, dehydroascorbic acid; GLP, germin like protein; Hepes, (N-[2-Hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]); IWF, intercellular washing fluid; OXO, oxalic acid oxidase (EC 1.2.3.4); PFD, photon flux density; TCA, trichloroacetic acid. * Corresponding author. Tel.: þ39 0763 374910; fax: þ39 0763 374980 E-mail addresses: [email protected] (S. Proietti), stefano.moscatello@ ibaf.cnr.it (S. Moscatello), [email protected] (F. Famiani), alberto.battistelli@ ibaf.cnr.it (A. Battistelli). 1 Tel.: þ39 0763 374906; fax: þ39 0763 374980. 2 Tel.: þ39 0763 374937; fax: þ39 0763 374980. 3 Tel.: þ39 075 585 6254; fax: þ39 075 585 6285. Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.03.010 Plant Physiology and Biochemistry 47 (2009) 717–723

Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

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lable at ScienceDirect

Plant Physiology and Biochemistry 47 (2009) 717–723

Contents lists avai

Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Increase of ascorbic acid content and nutritional quality in spinach leavesduring physiological acclimation to low temperature

Simona Proietti a,1, Stefano Moscatello a,2, Franco Famiani b,3, Alberto Battistelli a,*

a Istituto di Biologia Agroambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche (CNR), Viale Marconi 2, 05010 Porano (TR), Italyb Dipartimento di Scienze Agrarie e Ambientali, Universita degli Studi di Perugia –Borgo XX Giugno, 74, 06121 Perugia, Italy

a r t i c l e i n f o

Article history:Received 23 December 2008Accepted 19 March 2009Available online 28 March 2009

Keywords:AcclimationAscorbic acidOxalic acidLow temperatureSpinach

Abbreviations: AsA, ascorbic acid; DAsA, dehydroaprotein; Hepes, (N-[2-Hydroxyethyl]piperazine-N0-[2intercellular washing fluid; OXO, oxalic acid oxidasedensity; TCA, trichloroacetic acid.

* Corresponding author. Tel.: þ39 0763 374910; faxE-mail addresses: [email protected] (S. P

ibaf.cnr.it (S. Moscatello), [email protected] (F. Fibaf.cnr.it (A. Battistelli).

1 Tel.: þ39 0763 374906; fax: þ39 0763 374980.2 Tel.: þ39 0763 374937; fax: þ39 0763 374980.3 Tel.: þ39 075 585 6254; fax: þ39 075 585 6285.

0981-9428/$ – see front matter � 2009 Elsevier Masdoi:10.1016/j.plaphy.2009.03.010

a b s t r a c t

The effect of acclimation to 10 �C on the leaf content of ascorbic and oxalic acids, was investigated inspinach (Spinacia oleracea L.). At 10 �C the content of ascorbic acid in leaves increased and after 7 days itwas about 41% higher than in plants remaining under a 25 �C/20 �C day/night temperature regime. Incontrast, the content of oxalate, remained unchanged. Transfer to 10 �C increased the ascorbic but not theoxalic acid content of the leaf intercellular washing fluid (IWF). Oxalate oxidase (OXO EC 1.2.3.4) activitywas not detected in extracts of leaf blades. Therefore, oxalic acid degradation via OXO was not involved inthe control of its content. Our results show that low temperature acclimation increases nutritionalquality of spinach leaves via a physiological rise of ascorbic acid that does not feed-forward on thecontent of oxalic acid.

� 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction

Plant foods provide humans with most of their energetic andnutritional needs but can also be the major source of nutritionallynegative compounds. Ascorbic and oxalic acids are two relatedplant metabolites with opposite nutritional value for humans.While ascorbic acid (vitamin C) intake with plant food is highlypositive and can fulfil the daily intake need for humans [27], solubleand insoluble oxalate in the human and animal diet poses nutri-tional and health problems [37]. Soluble oxalic acid present in foodchelates cations in the digestive trait and this decreases theirintestinal absorption. High oxalic acid in the blood, that mightdepend on food intake, increases the risk of kidney stones [37],a large proportion of which are composed of calcium oxalate.Ascorbic acid present in food is thought to partially counteract thisnegative effect of oxalic acid [37].

scorbic acid; GLP, germin like-ethanesulfonic acid]); IWF,

(EC 1.2.3.4); PFD, photon flux

: þ39 0763 374980roietti), stefano.moscatello@amiani), alberto.battistelli@

son SAS. All rights reserved.

Ascorbic acid is present in all plant species and tissues where itplays a crucial role in energy metabolism, growth, development,response to biotic and a-biotic stresses and flowering [3]. Ascorbicacid can be synthesised by multiple pathways, actively studiedworld wide, and its turnover can be very fast, although relativelylittle is known about the contribution of ascorbic acid degradationto the control of ascorbic acid content of plant tissues [7] particu-larly under environmental stress conditions.

Oxalic acid is a widespread metabolite in the plant kingdom. Itcan be found as a soluble acid or insoluble salt, mainly as calciumoxalate. Considerable variations occur in the distribution of solubleand insoluble oxalate in plants, depending on the species, variety,age, tissues, [21,26] growth environment [33] and agriculturalpractices [40]. Several important roles for the physiology of theplant are now attributed to oxalic acid and to calcium oxalate.Oxalic acid is involved in the cellular pH stat [26], and it has beenconnected to nitrate reduction [1] and also involved in the oxidativereactions in the apoplast after pathogen invasion [19]. Insolublecalcium oxalate plays a role in osmoregulation [23,28] in defenceagainst grazing animals, in the detoxification of heavy metals (andoxalic acid). It also affects light distribution in leaves [26,27] and itis the product of excess calcium sequestration in idioblast [9,22].Oxalic acid in plant can be synthesised by different pathways [9],but the oxidative degradation of ascorbate that involves cleavage ofthe C2–C3 bond of the L-ascorbic acid carbon chain, is now indicatedas the most important one [22,23,27]. This is known to occur in thespecialised cells idioblasts, where the breakdown of ascorbic acidproduces the oxalic acid used for the formation of the calcium

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oxalate crystals [22,23,28]. The process of crystal formation ispromoted by calcium availability [29]. Crystal formation can bereversed by calcium starvation, perhaps with the involvement ofOXO [31]. In the case of idioblasts then, the increased synthesisof oxalic acid during crystal formation, drives the degradation ofascorbic acid and it is controlled by the need to sequester an excessof calcium as insoluble calcium oxalate [9]. Much less is knownabout the regulation of ascorbate-dependent synthesis of solubleoxalate in all other cell types, although soluble oxalate can be veryabundant in many plant tissues [10]. Loewus [27] pointed out thatfree radicals favour the cleavage of C2–C3 carbon bond of ascorbicacid to form oxalic acid and evidence has recently been provided foran apoplastic pathway of oxalate synthesis from ascorbate in Rosasuspension cells [14]. This pathway involves the conversion ofascorbate to L-threonate which is then converted to oxalate withthe involvement of an esterase. Even in this case, however, theconversion can also be achieved non-enzymatically. If and how thispathway is controlled is substantially unknown [14,26]. Guo andco-workers (2005) [15] have shown that feeding ascorbic acid or itsprecursor L-galactono g-lactone to rice seedlings causes an increaseof the ascorbic acid content that, beside being linked to an increasedresistance to chilling, causes a rise of the oxalate content, mainly ofits soluble form. Understanding the control of the metabolic linksbetween ascorbate and oxalate is of potentially great importance forimproving the nutritional quality of plant food, by increasingascorbate without a parallel increase of oxalic acid content.

To gain insight into spinach response to low temperatureacclimation under physiological conditions in term of nutritionalquality and into the control of ascorbic acid conversion to oxalicacid, we investigated whether the abundance of ascorbate/ascorbicacid and oxalate/oxalic acid was correlated in spinach leaves undera decrease of temperature in a physiological range. If oxalate isformed from ascorbate by non-enzymatic cleavage, an increase inascorbate content should feed-forward on the content of oxalate,unless export or catabolism of the latter is not increased corre-spondingly. We increased the ascorbate content of spinach leaves,by inducing plant acclimation to low growth temperature. Thecontent of ascorbate/ascorbic acid and oxalate/oxalic acid was thenmeasured in the symplast and apoplast. In addition, the abundanceof oxalate oxidase, the enzyme responsible for the catabolism ofoxalate was measured.

2. Results

Transfer of spinach plants from 25 �C to 10 �C caused a rapidacclimation of growth, photosynthesis and leaf photosynthetic endproducts turnover. Leaf dry weight and dry matter percentageincreased of about 30% and 28% respectively, while, specific leaffresh weight and chlorophyll contents were unchanged (Table 1).The transfer at low temperature rapidly and significantly affected

Table 1Leaf parameters. Characteristic of leaves of 7-weeks-old spinach plants that weregrown at 25 �C or were exposed for 7 days at 10 �C. Data are means of 6replicates � SE for growth parameters and of 4 replicates � SE for chlorophyllcontents. Statistical significance was tested by one-way ANOVA. When the F test wassignificant LSD between averages were calculated for P ¼ 0,05. For each variable,averages followed by different letters are statistically different; n.s. indicates that theF test was not significant.

Leaf parameters Temperature treatments

25 �C 10 �C

Specific leaf fresh weight (mg cm�2) 41.6 � 2.04 n.s. 43.4 � 0.95 n.s.Specific leaf dry weight (mg cm�2) 5.1 � 0.27 b 7.2 � 0.33 aDry matter (%) 11.9 � 0.31 b 16.4 � 0.51 aChlorophyll (mg cm�2) 25.0 � 4.00 n.s. 29.0 � 2.00 n.s.

gas exchange parameters. During the first day after transfer,assimilation rate (A) and stomatal conductance (gs) were respec-tively 36% and 75% lower at 10 �C than at 25 �C (values at 25 �C were23.8 m mol CO2 m�2 s�1 and 0.4 mol H2O m�2 s�1 for photosynthesisand stomatal conductance, respectively). Even if photosynthesiswas lower at 10 �C than at 25 �C, the content of photosynthetic endproducts in leaves increased. Low temperature acclimated leavescontained a significant larger pool of starch (Fig. 1B) and abouttwice the amount of sucrose (Fig. 1A), whereas, hexose contentswere not affected (data not shown). In leaves of plants grown forseven days at 10 �C, the amounts of soluble (glucose, fructose andsucrose (Fig. 1C)) and total (soluble þ starch (Fig. 1D)) non-struc-tural carbohydrate were respectively 51% and 46% higher than incontrol leaves.

Transfer to low growth temperature changed the leaf contentand distribution of ascorbic acid but not that of oxalic acid. Soonafter transfer to low temperature, bulk leaf content of ascorbicacid started to rise and reached its maximum after 172 h, witha 41% increase with respect to control leaves that during the sameperiod did not show significant changes in ascorbic acid content(Fig. 2A). During acclimation to low temperature the ratiobetween ascorbic and dehydroascorbic acid contents was alwaysvery high. Significant changes of the ratio were induced, inde-pendently from each other, by the time during the experimentand by the temperature treatment (insert in Figs. 2A and 3A).The ratio increased at the beginning of the experiment from 4 to28 h and decreased at the end of it from 100 to 172 h. At lowtemperature the ratio between ascorbate and dehydroascorbateincreased slightly although significantly, and, as an average, overthe entire duration of the experiment it was 93% and 95% for25 �C and 10 �C leaves. Acclimation to low temperature did notaffect the bulk leaf oxalic acid content that was also not affectedby the age of leaves nor by the combination of the two factors(Fig. 2B). More than 75% of the oxalic acid found in spinach leavesin this experiment was in the soluble form, this distribution wasnot affected by treatments (data not shown). Contents of ascorbicacid and oxalic acid in the leaf washing fluid were low, repre-senting on average only 2% and 0.2% of the bulk leaf content forascorbic and oxalic acid, respectively. The distribution of ascorbicacid between the symplast and the apoplast, but not that of oxalicacid, was affected by low growth temperature that increased fourtimes the apoplastic content of ascorbic acid (Fig. 3A and B). Theratio of ascorbic/oxalic acid in the apoplast was significantlyhigher than in leaves and clearly increased after acclimation tolow temperature (Compare the ratio in Fig. 3C with the absolutevalues in Fig. 2A and B).

The catabolic enzyme of oxalic acid, oxalic acid oxidase showedlarge variations in activity in different spinach tissues but was notdetected in the leaf blade under either normal or low growthtemperatures. Oxalate oxidase activity was highest in roots(1487 m mol H2O2 h�1 g fw�1) still high in the main leaf vein(73 m mol H2O2 h�1 g fw�1) but was not detectable in the leaf bladewhen the main vein was excluded. The high recovery of root OXOactivity in leaves (see Methods). demonstrates that the absence ofOXO activity in the leaf blade was not due to degradation of activityduring the measurement in this tissue.

3. Discussion

The interaction between the plant and its growing environmentcan profoundly affect the nutritional quality of food vegetables.Understanding how this interaction involves the control of relevantmetabolic pathways would allow a correct selection of the growingenvironment and can guide the genetic improvement of the specieby both traditional breeding and biotechnology. We show here that,

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S. Proietti et al. / Plant Physiology and Biochemistry 47 (2009) 717–723 719

in the case of spinach, a world wide diffused vegetable, acclimationof the plant to low growing temperature, under fully physiologicalconditions, causes a relevant increase in its quality, with respect toboth organoleptic and nutritional factors such as the leaf specificdry weight, the leaf content of non-structural carbohydrates andascorbic acid, without causing an increase of the nutritionalnegative metabolite oxalic acid.

Plants exposed to a decrease of growth temperature under highlight have to counteract an increased production of oxygen reactivespecies, to avoid oxidative damage of cell components [11]. Spinach iswell known to acclimate at low temperature by a profound modifi-cation of leaf metabolism. This includes an increase of photosyntheticenzymes [17] a light dependent modification of carbohydrate statusand leaf characteristics [4] and an increase of the capacity to scavengeoxygen reactive species [36]. The latter process involves specificallyan increase of ascorbic acid content in the chloroplast and even moremarkedly in the leaf blade [36].

Under our growing conditions spinach underwent a fullyphysiological acclimation of leaf metabolism to a decrease ingrowing temperature, as shown by data on leaf carbohydrate statuson the rise of ascorbic acid, and on the ratio between AsA and DAsA.We took advantage of this ability of spinach to obtain a relativelyrapid increase of the leaf ascorbic acid content under potentiallyoxidative conditions, to evaluate if the increase of the ascorbic acidcontent would feed-forward on the content of oxalic acid.

The rise of ascorbic acid did not feed-forward on the content ofoxalic acid, during low temperature acclimation of spinach plants.Oxalic acid can be synthesised from many metabolites in the plantcells (15 and references therein). More recently, however, a special,

and possibly prevalent role for ascorbic acid, in the synthesis ofoxalic acid was established. For example, in idioblasts an increase ofcalcium availability can switch on the deposition of calcium oxalatecrystals. The synthesis of oxalic acid used in the process, is fuelledwith ascorbic acid inside the idioblast [9,23]. In Medicago truncatulamutants overproducing oxalate crystals, Nakata and McConn(2007) [30] have found that overproduction of oxalate crystals iscoupled to a reduction of ascorbate content. However, many plantspecies, like spinach, accumulate large quantities of soluble oxalicacid, which is not used for the scavenging of excess calcium and it isnot confined to the specialised idioblasts cells. In our experimentssoluble oxalic acid was abundant and accounted invariably for morethan 75% of the total oxalate content of leaves, in accordance withvalues found by Savage (2000) [35] on the same species. It is likelythat the control of the synthesis of this large pool of oxalic acid isdifferent from that in idioblasts [9]. Green and Fry [14] have foundthat AsA can be converted to oxalic acid in the apoplast of Rosa cellsuspension via enzymatic and non-enzymatic degradation, aspreviously indicated by Loewus [27]. An un-controlled equilibriumbetween the two pools would cause the oxalic acid content ofleaves to rise when the AsA content is increased. Proietti et al. [33]showed that the pools of ascorbic and oxalic acid can respond in theopposite way when spinach plants are grown under different lightregimes. However this was obtained under a long time scaleexperiment and might not be indicative of relationships betweenthe two pools at the metabolic time scale. Guo and co-workers(2005) [15] induced an increase of oxalic acid content in riceseedlings by feeding both ascorbic acid and its precursor L-galactono g-lactone showing that an increase of the content of

Page 4: Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

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S. Proietti et al. / Plant Physiology and Biochemistry 47 (2009) 717–723720

oxalic acid, and particularly its soluble form, could be triggered byan increase of ascorbic acid in days time scale. However, this is notalways the case, as Li and Peng (2006) [25] did not obtain anincrease of oxalic acid after feeding roots of rice and buckwheatwith ascorbic acid. This demonstrates that the metabolic linkbetween ascorbic and oxalic acid might be controlled in differentways depending on the plant specie, tissue and possibly environ-mental conditions.

In the present study there was no rise of oxalic acid in spinachleaves, although we caused a relevant rise of AsA in a relativelyshort time (7 days) under conditions that favour the formation offree radicals. This shows that, in the largely diffused food vegetablespinach, the pools of ascorbic and oxalic acids were not linked bya simple chemical equilibrium and indicates that the conversion ofascorbic to oxalic acid is under tight control even in the relativelyshort time span of our experiment. One of the reasons could be thatspinach was perfectly able to cope with the oxidative threateningdue to low temperature as show by the AsA/DAsA ratio that washigher in low temperature than in the control leaves. This isparticularly important for a leafy food vegetable like spinach,because increasing its nutritional quality by increasing the contentof ascorbic acid would be unfeasible if linked to an increase of thenutritionally negative metabolite oxalic acid.

The control of exchanges between two metabolite pools canbe partially exerted by metabolic compartmentation. However,ascorbic acid is known to be present in many cellular compart-ments [18]. Schoner and Krause [36] found that the rise of ascorbic

acid under acclimation to low temperature, was not confined tochloroplasts, where most of the oxygen reactive species areproduced, but was spread over other cellular compartments sinceits content in the bulk leaf lamina increased much more than inchloroplasts. Likewise, in both our experiments, the ascorbic acidcontent and the ascorbic/oxalic acid ratio increased in the apoplastafter acclimation to 10 �C. This shows that the burst of ascorbic acid

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S. Proietti et al. / Plant Physiology and Biochemistry 47 (2009) 717–723 721

caused by low temperature acclimation was not confined to specificcellular compartments. In particular it reached the apoplast, wherethe Green and Fry [14] ascorbic acid degradation pathway islocated, making it unlikely that compartmentation would limit theascorbic acid dependent synthesis of oxalic acid.

Another site of control of the abundance of one metabolite ina specific tissue could be its export. It is unlikely that export fromleaves is responsible for keeping the oxalic acid content low withrespect to that of the ascorbic acid. Firstly because phloem exportfrom leaves at low temperature is strongly reduced as shown by ourcarbohydrate data and as reported by Strand et al. [38] andsecondly because the mobility of oxalic acid in leaves is limited tolocal diffusion, while ascorbic acid is loaded in phloem for longdistance transport [10].

Degradation of one metabolite can indeed contribute to keepits concentration low in the presence of active synthesis. Oxalicacid can be degraded to CO2 and H2O2 by the enzyme oxalic acidoxidase. Oxalic acid degradation via OXO can then maintaina low content of oxalic acid even in the presence of an activesynthesis. For example, rapid oxalic acid metabolism was foundafter feeding 14C oxalic acid to tobacco leaf discs [16]. OXOactivity is present in several organisms ranging from bacteria[24] to fungi and higher plants [12]. In cereals, OXO activityis almost constitutively associated to germin proteins [5],frequently in the apoplastic cell compartment [8]. Our resultsindicate that it is unlikely that spinach leaf blade undergoesrapid oxalic acid degradation via OXO activity under normaltemperature nor under 10 �C acclimation.

In conclusion, acclimation of spinach plants to growth at 10 �Ccaused an increase of its nutritional quality that included a fullyphysiological increase of ascorbic acid that did not feed-forward onthat of oxalic acid. Compartmentation of ascorbic acid in the plantcell, export to other tissues and degradation via OXO activity can allbe reasonably discarded as causes of this lack of equilibriumbetween the two pools. Our results suggest, although direct andconclusive evidences should be obtained by further investigations,that the non-enzymatic conversion of ascorbic acid to oxalic acid isunlikely and that the ascorbic acid dependent synthesis of solubleoxalic seems to be under tight control, at least in spinach duringacclimation to low growth temperature. This would give thepossibility to breeders to increase the spinach content of ascorbicacid in order to improve its the nutritional quality. It also highlightsthe importance of environmental growing conditions in controllingthe nutritional quality of plant foods.

4. Methods

4.1. Chemicals

4-aminoantipyrine, ascorbic acid, 2,2’dipyridyl, DL-dithio-threitol (DTT), ferric chloride (FeCl3), hydrogen peroxide (H2O2), N-[2-Hydroxyethyl]piperazine-N0-[2-ethanesulfonic acid] (Hepes),magnesium chloride (MgCl2), N,N’dimethylanilin, N-ethyl-maleimide, oxalic acid, potassium chloride (KCl), potassiumphosphate dibasic (K2HPO4), sodium hydroxide (NaOH), sodiumacetate, sodium succinate, trichloroacetic acid (TCA), albumin frombovine serum (BSA), phosphoglucose isomerase, invertase werefrom Sigma (St. Louis, MO). Ethanol, amyloglucosidase, from Fluka(Sigma), ortho-phosphoric acid (H3PO4) were from Merck (MerckKGaA, Germany), hydrochloric acid (HCl) from Panreac (BarcelonaSpain), potassium hydroxide (KOH) from BDH Chemicals (LtdPoole England). Hexokinase, glucose phosphate dehydrogenase,a-amylase were from Roche (Mannheim Germany). Oxalic acid wasmeasured by a test combination from r-biopharm (Roche, Man-nheim Germany).

4.2. Plant material and growth conditions

Spinach (cv. Gigante invernale) were sown, and thinned to twoplants, in 1 l plastic pots, containing a mixture of soil: sand: perlite(2:1:1, v/v). Plants were grown in growth cabinets (Fitotron SGD170Sanyo Gallenkamp U.K.) under a 10 h light/14 h dark photoperiod.Photon flux density (PFD l 400–700 nm) was 800 � 5% mmolquanta m�2 s�1, while relative humidity was 70% � 5% and CO2

concentration 360 ppm � 10 ppm. Temperature was 25 � 0.5 �Cand 20 � 0.5 �C for the light and dark period respectively. Duringthe growth, plants were fed twice a week with a full nutrientsolution and supplied with water as required. Acclimation to lowtemperature started 35 days after germination, by moving half ofthe pots to a twin cabinet, set with a day/night temperature regimeof 10 � 0.5 �C/10 � 0.5 �C.

4.3. Sampling and analysis

Measurements of gas exchange were made at PFD of 800 mmolquanta m�2 s�1 on the fourth fully expanded leaves, at the growingtemperature, 4 and 28 h after the onset of acclimation, usinga portable open gas exchange system (Li-6400, LI-COR, Lincoln, NE,USA). The specific leaf fresh weight (fresh weight/leaf area) and thespecific leaf dry weight (dry weight/leaf area) were measured on leafdiscs of 4 cm2 cut from the lamina with a sharp cork borer. Dryweight was obtained by freeze drying leaves to constant weight. Allchemical analysis was performed on the fourth and fifth fullyexpanded leaves.

4.4. Leaf carbohydrate analysis

Measurements of non-structural carbohydrate were performedon leaf discs, 1 cm2, collected from the leaf lamina with a cork borerand immediately frozen in liquid nitrogen. Frozen discs wereground in a glass–glass homogenizer with 1.5 ml of 80% (v/v)ethanol, 20% (v/v) 100 mM Hepes (pH 7.3), 10 mM MgCl2 andextracted at 80 �C for 45 min. The extract was centrifuged at12 000 g for 5 min, soluble sugars (glucose, fructose and sucrose)were recovered in the supernatant, starch was in the pellet. Solublesugars determination, by spectrophotometric coupled enzymaticassay, was performed as in Antognozzi et al. [2]. The carbohydrateassay medium contained 100 mM Hepes pH 7.1, 10 mM MgCl2,0.5 mM DTT, 0.01 BSA (w/v), 100 mM ATP, and 80 mM NADþ and20 nkat of hexokinase. Shortly, the ethanol extract (10–20 ml ina total volume of 200 ml) was added to the assay solution, causingglucose and fructose to be phosphorylated to glucose-6 P andfructose-6 P by the hexokinase. After adding 5 nkat of glucosephosphate dehydrogenase, glucose is oxidised to 6-phosphogluconate with the stoichiometric production of NADH which ismeasured, after the reaction has reached the end point, as thechange in absorbance at 340 nm (extinction coefficient ¼6.23 mM cm�1). After reaching the end point for glucosemeasurement, 5 nkat of phosphoglucose isomerase were added,causing the isomerisation of fructose-6 P to glucose-6 P thateventually entered the previously described reaction causinga second rise of the NADH concentration. After the fructose reading,adding of 500 nkat of invertase, hydrolysed sucrose to glucose andfructose that were phosphorylated and entered the previouslydescribed reaction causing a third increase of NADH (two moleculesof NADH produced for each sucrose molecule present in theextract). The pellet, containing starch, was washed three timeswith 50 mM NaAcetate buffer (pH 4.5) and then suspended andautoclaved at 120 �C for 45 min in 1 ml of the same buffer. Afterautoclaving, the sample was incubated at 50 �C for 1 h with

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amyloglucosidase (668 nkat) and a-amilase (66.8 nkat) to hydro-lyse starch to glucose, that was then measured as described before.

Chlorophylls were measured spectrophotometrically, using theethanolic extract of carbohydrate as in Graan and Hort [13].

4.5. Determination of ascorbate (AsA) and dehydroascobate (DasA)

Ascorbic acid content was determined on leaf discs of 2.5 cm2

taken, one per plant, with a cork borer, immediately frozen in liquidN2, and there stored until required. Discs were ground usinga glass–glass homogenizer containing 1.5 ml of 10% (w/v) TCA, andcentrifuged at 12 000 g for 15 min. AsA, and DAsA in the super-natant were measured using a colorimetric assay [20]. Totalascorbate (AsA þ DasA) was determined through a reduction ofDAsA to AsA by 2 mM DTT. For AsA measurement, the assay solu-tion (1 ml final volume) contained 0.1 ml of leaf extract, 2.5% TCA,0.8% 2,2’dipyridyl, 0.3% FeCl3 reading of absorbance was made at525 nm. For the total ascorbate (AsA þ DasA) measurement theassay mixture (1 ml final volume), containing 0.1 ml of leaf extract,2 mM DTT and 0.1% N-ethylmaleimide, was incubated at 42 �C for15 min. After reduction of DAsA to AsA the color was developed byadding 2.5% TCA, 0.8% 2,2’dipyridyl, 0.3% FeCl3 and the absorbancewas measured at 525 nm. For each sample, DAsA was calculated asthe difference between total ascorbate and reduced ascorbateconcentrations. A standard curve in the range of 0–80 nmolascorbate was used.

4.6. Determination of oxalic acid

For the determination of oxalic acid leaf discs of 2.5 cm2, wereextracted using a glass–glass homogenizer containing 1.5 ml ofdistilled water. The extract was centrifuged at 12 000 g for 5 min at4 �C. The supernatant was recovered for measurements of thesoluble oxalic acid, the pellet was washed once with 1 ml of distilledwater and re-suspended in 1.5 ml of water. After rapid adjusting ofthe pH to 2.8 with 1 N HCl, the suspension was placed at 50 �C for15 min for complete solubilization of oxalate salts, then the pH wasadjusted to 5.0. Oxalic acid was measured using the enzymatic assaydescribed by Beutler et al. [6]. Oxalate was cleaved to formic acidand CO2 at pH 5.0 in presence of oxalate decarboxylase. The formicacid formed was quantitatively oxidized to bicarbonate by NADþ atpH 7.5 in the presence of the enzyme formate dehydrogenase. Theamount of NADH formed during the last reaction was measured asthe change in absorbance at 340 nm.

4.7. Determination of oxalic acid and ascorbic acid in the apoplast

Ascorbate and oxalate in the apoplast were measured in theintercellular washed fluid obtained as described by Pasqualini et al.[32]. Shortly, spinach leaf discs of 7 cm2 were washed with distilledwater and vacuum infiltrated with a solution of KCl 100 mM pH 5.5using 1 g of leaves in 100 ml of solution. The leaf material was thencarefully placed in plastic syringes, centrifuged at 1500 g for 5 mina 4 �C and the solution obtained was used for metabolitemeasurements.

4.8. Oxalate oxidase assay

Oxalate oxidase (OXO; EC 1.2.3.4) cleaves one molecule of oxalicacid (þO2 and 2Hþ) to H2O2 and 2CO2. The OXO activity and wasmeasured in the same leaves used for metabolites quantification, bythe colorimetric assay described by Zhang et al. [39] with somemodifications. Fresh leaves were ground in a glass–glass homoge-nizer containing distilled water (1:10 w/v) and after centrifugationat 5000 g for 8 min, the supernatant and pellet were checked for

OXO activity. The reaction solution (1 ml final volume), contained50 mM sodium succinate pH 3.8, 5 mM oxalic acid, 20 ml/100 mlN,N’dimethylanilin, 8 mg/100 ml 4-aminoantipyrine, and 33.4 nkatml�1 of horseradish peroxydase. The reaction was started by addingthe sample, and after incubation at 37 �C for 20 min, it was stoppedby adding 20 ml NaOH 1 M. The change in absorbance was measuredat 550 nm. The H2O2 produced was estimated by comparison witha standard curve in the range of 0–50 nmol.

4.9. Equipments, recovery and statistical analysis

Carbohydrate and oxalic acid analysis were made with an Elisaplate reader (Anthos, 2001; Anthos Labtec Instruments, Salzburg,Austria) equipped with a kinetic software that allowed the record ofthe enzymatic reactions by reading absorbance changes every 15 sand that allowed the calculation of the absorbance differencebetween the start and the end of the reaction. Instrument internalerror was �1 milli absorbance unit, assays were adjusted to havereadings at least 100 times higher than the instrument’s internalerror. All measurements of a single sample, were made in duplicate,if the two measurements differed for more than 5% of the reading,the measurement was repeated. Recovery of carbohydrate, for thewhole procedure, was always higher than 90%. The recovery ofsoluble and insoluble oxalate was tested for the whole procedure[34] and it was always close to 100 with no effects of the procedureon the soluble/insoluble ratio. Chlorophyll, ascorbic acid and OXOactivities were measured with a SIGMA ZWS11 spectrophotometer(ZWS 11 SIGMA BIOCHEM, Puchheim, Germany) in the singlewavelength mode, the instrument has internal error lower than1 milli absorbance unit, the assays were arranged in order to havereadings at least 100 times higher than the instrument’s internalerror. Recovery tests for ascorbic acid analysis gave recovery close to95%. A recovery test of OXO activity in leaves was performed usingroot tissue that showed high OXO activity, the recovery was alwaysclose to 100%.

Statistical analysis was performed by ANOVA using the STATIS-TICA software package (StatSoft for Windows, 1998). Variableswere analysed by one way or two ways ANOVA as required by theexperimental design, refer to table and figure legends for details.When the F test was significant, differences between averages weretested by an LSD with P ¼ 0.05.

Acknowledgements

The authors are indebted to Dr. Robert P. Walker for criticalreading of the manuscript. This work was partially supported by theItalian National Research Council contract RTL no 010.08.M001.

References

[1] A.K. Ahmed, K.A. Johnson, The effect of the ammonium: nitrate nitrogen ratio,total nitrogen, salinity (NaCl) and calcium on the oxalate levels of Tetragoniatetragonioides Pallas. Kunz, J. Hort. Sci. Biotechnol. 75 (2000) 533–538.

[2] E. Antognozzi, A. Battistelli, F. Famiani, S. Moscatello, F. Stanica, A. Tombesi,Influence of CPPU on carbohydrate accumulation and metabolism in fruits ofActinidia deliciosa (A. Chev.), Sci. Hort 65 (1996) 37–47.

[3] C. Barth, M. De Tullio, P.L. Conklin, The role of ascorbic acid in the control offlowering time and the onset of senescence, J. Exp. Bot. 57 (2006) 1657–1665.

[4] A. Battistelli, W. Martindale, R.C. Leegood, in: P. Mathis (Ed.), Effects of Lightand Carbohydrate Content on Acclimation of Spinach Photosynthesis to LowTemperature, Photosynthesis: from Light to Biosphere, vol. IV, KluwerAcademic Publishers, 1995, pp. 865–868.

[5] F. Bernier, A. Berna, Germin and germin-like proteins: plant do-all proteins.But what do they do exactly? Plant Physiol. Biochem. 39 (2001) 545–554.

[6] H.O. Beutler, J. Becker, G. Michal, E. Walter, Rapid method for determination ofOxalate, Fresenius Zeitschrift fur Analytische Chemie 301 (1980) 186–187.

[7] S. Debolt, V. Melino, C.M. Ford, Ascorbate as a biosynthetic precursor in plants,Ann. Bot. 99 (2007) 3–8.

Page 7: Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

S. Proietti et al. / Plant Physiology and Biochemistry 47 (2009) 717–723 723

[8] J.M. Dunwell, S. Khuri, P.J. Gane, Microbial relatives of the see storageproteins of higher plants: conservation of structure and diversification offunctions during evolution of the cupin superfamily, Microbiol. Mol. Biol. Rev.64 (2000) 153–179.

[9] V. Franceschi, P.A. Nakata, Calcium oxalate in plants: formation and function,Annu. Rev. Plant. Biol. 56 (2005) 41–71.

[10] V.R. Franceschi, N.M. Tarlyn, L-ascorbic acid is accumulated in source leafphloem and transported to sink tissues in plants, Plant Physiol. 130 (2002)649–656.

[11] L.D. Gomez, H. Vanaker, P. Buchner, G. Noctor, C.H. Foyer, Intercellular distri-bution of glutathione synthesis in maize leaves and its response to short-termchilling, Plant Physiol. 134 (2004) 1662–1671.

[12] L. Goyal, M. Thakur, C.S. Pundir, Purification and properties of a membrane boundoxalate oxidase from Amaranthus leaves, Plant Sci. 142 (1999) 21–28.

[13] T. Graan, D.R. Hort, Quantitation of the rapid electron donar to P700, thefunctional plastoquinone pool and the ratio of photosystem in spinach clor-oplast, J. Biol. Chem. 259 (1984) 14003–14010.

[14] M.A. Green, S.C. Fry, Vitamin C degradation in plant cell via enzymatichydrolysis of 4-0-oxalyl-L-threonate, Nature 433 (2005) 83–87.

[15] Z. Guo, H. Tan, Z. Zhu, S. Lu, B. Zhou, Effect of intermediates on ascorbic acidand oxalate biosynthesis of rice and in relation to its stress resistance, PlantPhysiol. Biochem. 43 (2005) 955–962.

[16] E.A. Havir, Oxalate metabolism by tobacco leaf discs, Plant Physiol. 75 (1984)505–507.

[17] A.S. Holaday, W. Martindale, R. Alred, A. Brooks, R.C. Leegood, Changes inactivities of enzymes of carbon metabolism in leaves during exposure to lowtemperature, Plant Physiol. 98 (1992) 1105–1114.

[18] N. Horemans, C.H. Foyer, G. Potters, H. Asard, Ascorbate function and associ-ated transport systems in plants, Plant. Physiol. Biochem. 38 (2000) 531–540.

[19] X. Hu, D.L. Bidney, N. Yalpani, J.P. Duvick, O. Crasta, O. Folkerts, G.H. Lu, Over-expression of a gene encoding hydrogen peroxide-generating oxalate oxidaseevokes defense responses in sunflower, Plant Physiol. 133 (2003) 170–181.

[20] K. Kampfenkel, M. van Montagu, D. Inze, Extraction and determination of ascorbateand dehydroascorbate from plant tissues, Anal. Biochem. 225 (1995) 165–216.

[21] Y. Kavazu, M. Okimura, T. Ishii, S. Yui, Varietal and seasonal differences inoxalate content of spinach, Sci. Hort. 97 (2003) 203–210.

[22] S.E. Keates, N.M. Tarlyn, F.A. Loewus, V.R. Franceschi, L-Ascorbic acid andL-galactose are sources for oxalic acid and calcium oxalate in Pistia stratiotes,Phytochemistry 53 (2000) 433–440.

[23] T.A. Kostman, N.M. Tarlyn, F.A. Loewus, V.R. Franceschi, Biosynthesis ofL-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalicacid occurs within individual calcium oxalate crystal idioblasts, Plant. Physiol.125 (2001) 634–640.

[24] H. Koyama, Purification and characterization of oxalate oxidase from Pseu-domonas sp. OX-53, Agric. Biol. Chem. 52 (1988) 743–748.

[25] B.S. Li, X.X. Peng, Relationship between oxalate accumulation and ascorbatocontent in plant leaves, Plant Physiol. Commun. 42 (2006) 31–33.

[26] B. Libert, V.R. Franceschi, Oxalate in crop plants, J. Agri. Food. Chem. 35 (1987)926–938.

[27] F.A. Loewus, Biosynthesis and metabolism of ascorbic acid in plants and ofanalogs of ascorbic acid in fungi, Phytochemistry 52 (1999) 447–457.

[28] A.M.A. Mazen, D. Zhang, V.R. Franceschi, Calcium oxalate formation in Lemnaminor: physiological and ultrastructure aspects of high capacity calciumsequestration, New. Phytol. 161 (2003) 435–448.

[29] P.A. Nakata, Advances in our understanding of calcium oxalate crystalformation and function in plants, Plant. Sci. 164 (2003) 901–909.

[30] P.A. Nakata, M. McConn, Isolated Medicago truncatula mutants with increasedcalcium oxalate crystal accumulation have decreased ascorbic acid levels,Plant Physiol. Biochem. 45 (2007) 216–220.

[31] E.A.S. Paiva, S.R. Machado, Role of intermediary cells in Peltodon radicans(Laminiaceae) in the transfer of calcium and formation of calcium oxalatecrystals, Brazilian. Arch. Biotechnol. 48 (2005) 147–153.

[32] S. Pasqualini, G. Della Torre, F. Ferranti, L. Ederli, C. Piccioni, L. Reale,M. Antonielli, Salicylic acid modulates ozone-induced hypersensitive celldeath in tobacco plants, Physiol. Plantarum 115 (2002) 204–212.

[33] S. Proietti, S. Moscatello, A. Leccese, G. Colla, A. Battistelli, The effect ofgrowing spinach (Spinacia oleracea L.) at two light intensities on the amountsof oxalate, ascorbate and nitrate in their leaves, J. Hort. Sci. Biotechnol. 79(2004) 606–609.

[34] M. Rassam, W. Laing, Variation in ascorbic acid and oxalate level in the fruitof Actinidia chinensis tissues and genotypes, J. Agric. Food Chem. 53 (2005)2322–2326.

[35] G.P. Savage, L. Vanhanen, S.M. Mason, A.B. Ross, Effect of cooking on thesoluble and insoluble oxalate content of some New Zealand foods, J. Food.Comp. An. 13 (2000) 201–206.

[36] S. Schoner, G.H. Krause, Protective systems against active oxygen species inspinach: response to cold acclimation in excess light, Planta 180 (1990) 383–389.

[37] R. Siener, D. Ebert, C. Nicolay, A. Hesse, Dietary risk factors for hyperoxaluria incalcium oxalate stone formers, Kid. Intern. 63 (2003) 1037–1043.

[38] Å. Strand, V. Hurry, S. Henkes, N. Huner, P. Gustafsson, P. Gardestrom, M. Stitt,Acclimation of Arabidopsis leaves developing at low temperatures. Increasingcytoplasmic volume accompanies increase activities of enzymes in the Calvincycle and in the sucrose-biosynthetic pathway, Plant Physiol. 119 (1999)1387–1397.

[39] Z.G. Zhang, J. Yang, D.B. Collinge, H. Thordal-Christensen, Ethanol increasessensitivity of oxalate oxidase assays and facilitates direct activity staining inSDS gels, Plant Mol. Biol.Rep. 14 (1996) 266–272.

[40] H. Zimmerman, The influence of fertilization on the quality of spinach atvarious light intensitiesSymposium of vegetable growing under glass, ActaHort. 4 (1996) 89–95.