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

Plant Physiology and Biochemistry 47 (2009) 717–723

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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 b

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

a r t i c l e i n f o

a b s t r a c t

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

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.

Keywords: Acclimation Ascorbic acid Oxalic acid Low temperature Spinach

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 nutritional 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].

Abbreviations: AsA, ascorbic acid; DAsA, dehydroascorbic acid; GLP, germin like protein; Hepes, (N-[2-Hydroxyethyl]piperazine-N0 -[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. 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.03.010

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] particularly 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 C2–C3 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

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oxalate crystals [22,23,28]. The process of crystal formation is promoted by calcium availability [29]. Crystal formation can be reversed by calcium starvation, perhaps with the involvement of OXO [31]. In the case of idioblasts then, the increased synthesis of oxalic acid during crystal formation, drives the degradation of ascorbic acid and it is controlled by the need to sequester an excess of calcium as insoluble calcium oxalate [9]. Much less is known about the regulation of ascorbate-dependent synthesis of soluble oxalate in all other cell types, although soluble oxalate can be very abundant in many plant tissues [10]. Loewus [27] pointed out that free radicals favour the cleavage of C2–C3 carbon bond of ascorbic acid to form oxalic acid and evidence has recently been provided for an apoplastic pathway of oxalate synthesis from ascorbate in Rosa suspension cells [14]. This pathway involves the conversion of ascorbate to L-threonate which is then converted to oxalate with the involvement of an esterase. Even in this case, however, the conversion can also be achieved non-enzymatically. If and how this pathway is controlled is substantially unknown [14,26]. Guo and co-workers (2005) [15] have shown that feeding ascorbic acid or its precursor L-galactono g-lactone to rice seedlings causes an increase of the ascorbic acid content that, beside being linked to an increased resistance to chilling, causes a rise of the oxalate content, mainly of its soluble form. Understanding the control of the metabolic links between ascorbate and oxalate is of potentially great importance for improving the nutritional quality of plant food, by increasing ascorbate without a parallel increase of oxalic acid content. To gain insight into spinach response to low temperature acclimation under physiological conditions in term of nutritional quality and into the control of ascorbic acid conversion to oxalic acid, we investigated whether the abundance of ascorbate/ascorbic acid and oxalate/oxalic acid was correlated in spinach leaves under a decrease of temperature in a physiological range. If oxalate is formed from ascorbate by non-enzymatic cleavage, an increase in ascorbate content should feed-forward on the content of oxalate, unless export or catabolism of the latter is not increased correspondingly. We increased the ascorbate content of spinach leaves, by inducing plant acclimation to low growth temperature. The content of ascorbate/ascorbic acid and oxalate/oxalic acid was then measured in the symplast and apoplast. In addition, the abundance of oxalate oxidase, the enzyme responsible for the catabolism of oxalate was measured. 2. Results Transfer of spinach plants from 25  C to 10  C caused a rapid acclimation of growth, photosynthesis and leaf photosynthetic end products turnover. Leaf dry weight and dry matter percentage increased of about 30% and 28% respectively, while, specific leaf fresh weight and chlorophyll contents were unchanged (Table 1). The transfer at low temperature rapidly and significantly affected

Table 1 Leaf parameters. Characteristic of leaves of 7-weeks-old spinach plants that were grown at 25  C or were exposed for 7 days at 10  C. Data are means of 6 replicates  SE for growth parameters and of 4 replicates  SE for chlorophyll contents. Statistical significance was tested by one-way ANOVA. When the F test was significant 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 the F test was not significant. Leaf parameters

Temperature treatments 25  C

Specific leaf fresh weight (mg cm2) Specific leaf dry weight (mg cm2) Dry matter (%) Chlorophyll (mg cm2)

41.6 5.1 11.9 25.0

   

10  C 2.04 0.27 0.31 4.00

n.s. b b n.s.

43.4 7.2 16.4 29.0

   

0.95 0.33 0.51 2.00

n.s. a a n.s.

gas exchange parameters. During the first day after transfer, assimilation rate (A) and stomatal conductance (gs) were respectively 36% and 75% lower at 10  C than at 25  C (values at 25  C were 23.8 m mol CO2 m2 s1 and 0.4 mol H2O m2 s1 for photosynthesis and stomatal conductance, respectively). Even if photosynthesis was lower at 10  C than at 25  C, the content of photosynthetic end products in leaves increased. Low temperature acclimated leaves contained a significant larger pool of starch (Fig. 1B) and about twice the amount of sucrose (Fig. 1A), whereas, hexose contents were not affected (data not shown). In leaves of plants grown for seven days at 10  C, the amounts of soluble (glucose, fructose and sucrose (Fig. 1C)) and total (soluble þ starch (Fig. 1D)) non-structural carbohydrate were respectively 51% and 46% higher than in control leaves. Transfer to low growth temperature changed the leaf content and distribution of ascorbic acid but not that of oxalic acid. Soon after transfer to low temperature, bulk leaf content of ascorbic acid started to rise and reached its maximum after 172 h, with a 41% increase with respect to control leaves that during the same period did not show significant changes in ascorbic acid content (Fig. 2A). During acclimation to low temperature the ratio between ascorbic and dehydroascorbic acid contents was always very high. Significant changes of the ratio were induced, independently from each other, by the time during the experiment and by the temperature treatment (insert in Figs. 2A and 3A). The ratio increased at the beginning of the experiment from 4 to 28 h and decreased at the end of it from 100 to 172 h. At low temperature the ratio between ascorbate and dehydroascorbate increased slightly although significantly, and, as an average, over the entire duration of the experiment it was 93% and 95% for 25  C and 10  C leaves. Acclimation to low temperature did not affect the bulk leaf oxalic acid content that was also not affected by the age of leaves nor by the combination of the two factors (Fig. 2B). More than 75% of the oxalic acid found in spinach leaves in this experiment was in the soluble form, this distribution was not affected by treatments (data not shown). Contents of ascorbic acid and oxalic acid in the leaf washing fluid were low, representing on average only 2% and 0.2% of the bulk leaf content for ascorbic and oxalic acid, respectively. The distribution of ascorbic acid between the symplast and the apoplast, but not that of oxalic acid, was affected by low growth temperature that increased four times the apoplastic content of ascorbic acid (Fig. 3A and B). The ratio of ascorbic/oxalic acid in the apoplast was significantly higher than in leaves and clearly increased after acclimation to low temperature (Compare the ratio in Fig. 3C with the absolute values in Fig. 2A and B). The catabolic enzyme of oxalic acid, oxalic acid oxidase showed large variations in activity in different spinach tissues but was not detected in the leaf blade under either normal or low growth temperatures. Oxalate oxidase activity was highest in roots (1487 m mol H2O2 h1 g fw1) still high in the main leaf vein (73 m mol H2O2 h1 g fw1) but was not detectable in the leaf blade when the main vein was excluded. The high recovery of root OXO activity in leaves (see Methods). demonstrates that the absence of OXO activity in the leaf blade was not due to degradation of activity during the measurement in this tissue. 3. Discussion The interaction between the plant and its growing environment can profoundly affect the nutritional quality of food vegetables. Understanding how this interaction involves the control of relevant metabolic pathways would allow a correct selection of the growing environment and can guide the genetic improvement of the specie by both traditional breeding and biotechnology. We show here that,

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(+ 52,4 %)

0 25 °C

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Fig. 1. Carbohydrate contents (hexose equivalents mmol g fw1) in leaves of 7-weeks-old spinach plants that were grown at 25  C or were exposed for 7 days at 10  C. Data are means of 4 replicates  SE. Statistical significance was tested by one-way ANOVA. When the F test was significant, LSD were calculated for P ¼ 0.05 and indicated by different letters.

in the case of spinach, a world wide diffused vegetable, acclimation of the plant to low growing temperature, under fully physiological conditions, causes a relevant increase in its quality, with respect to both organoleptic and nutritional factors such as the leaf specific dry weight, the leaf content of non-structural carbohydrates and ascorbic acid, without causing an increase of the nutritional negative metabolite oxalic acid. Plants exposed to a decrease of growth temperature under high light have to counteract an increased production of oxygen reactive species, to avoid oxidative damage of cell components [11]. Spinach is well known to acclimate at low temperature by a profound modification of leaf metabolism. This includes an increase of photosynthetic enzymes [17] a light dependent modification of carbohydrate status and leaf characteristics [4] and an increase of the capacity to scavenge oxygen reactive species [36]. The latter process involves specifically an increase of ascorbic acid content in the chloroplast and even more markedly in the leaf blade [36]. Under our growing conditions spinach underwent a fully physiological acclimation of leaf metabolism to a decrease in growing temperature, as shown by data on leaf carbohydrate status on the rise of ascorbic acid, and on the ratio between AsA and DAsA. We took advantage of this ability of spinach to obtain a relatively rapid increase of the leaf ascorbic acid content under potentially oxidative conditions, to evaluate if the increase of the ascorbic acid content would feed-forward on the content of oxalic acid. The rise of ascorbic acid did not feed-forward on the content of oxalic acid, during low temperature acclimation of spinach plants. Oxalic acid can be synthesised from many metabolites in the plant cells (15 and references therein). More recently, however, a special,

and possibly prevalent role for ascorbic acid, in the synthesis of oxalic acid was established. For example, in idioblasts an increase of calcium availability can switch on the deposition of calcium oxalate crystals. The synthesis of oxalic acid used in the process, is fuelled with ascorbic acid inside the idioblast [9,23]. In Medicago truncatula mutants overproducing oxalate crystals, Nakata and McConn (2007) [30] have found that overproduction of oxalate crystals is coupled to a reduction of ascorbate content. However, many plant species, like spinach, accumulate large quantities of soluble oxalic acid, which is not used for the scavenging of excess calcium and it is not confined to the specialised idioblasts cells. In our experiments soluble oxalic acid was abundant and accounted invariably for more than 75% of the total oxalate content of leaves, in accordance with values found by Savage (2000) [35] on the same species. It is likely that the control of the synthesis of this large pool of oxalic acid is different from that in idioblasts [9]. Green and Fry [14] have found that AsA can be converted to oxalic acid in the apoplast of Rosa cell suspension via enzymatic and non-enzymatic degradation, as previously indicated by Loewus [27]. An un-controlled equilibrium between the two pools would cause the oxalic acid content of leaves to rise when the AsA content is increased. Proietti et al. [33] showed that the pools of ascorbic and oxalic acid can respond in the opposite way when spinach plants are grown under different light regimes. However this was obtained under a long time scale experiment and might not be indicative of relationships between the two pools at the metabolic time scale. Guo and co-workers (2005) [15] induced an increase of oxalic acid content in rice seedlings by feeding both ascorbic acid and its precursor Lgalactono g-lactone showing that an increase of the content of

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1,5

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Control (25 °C) Chilling (10 °C) (93.5 b) (97.3 a)

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Fig. 2. Ascorbic acid (Panel A) and oxalic acid (Panel B) contents of total leaf lamina in spinach, during acclimation to 10  C. The F test (ANOVA) for the interaction TIME of 10  C exposure X TEMPERATURE was statistically significant for ascorbate content (Panel A), but not for oxalate content that was also not significantly affected separately by the main factors. Differences between averages were tested by LSD test for P ¼ 0.05 and are indicated by different letters on the top of each graph bar.

C 1,2

Ascorbic acid /oxalic acid

oxalic acid, and particularly its soluble form, could be triggered by an increase of ascorbic acid in days time scale. However, this is not always the case, as Li and Peng (2006) [25] did not obtain an increase of oxalic acid after feeding roots of rice and buckwheat with ascorbic acid. This demonstrates that the metabolic link between ascorbic and oxalic acid might be controlled in different ways depending on the plant specie, tissue and possibly environmental conditions. In the present study there was no rise of oxalic acid in spinach leaves, although we caused a relevant rise of AsA in a relatively short time (7 days) under conditions that favour the formation of free radicals. This shows that, in the largely diffused food vegetable spinach, the pools of ascorbic and oxalic acids were not linked by a simple chemical equilibrium and indicates that the conversion of ascorbic to oxalic acid is under tight control even in the relatively short time span of our experiment. One of the reasons could be that spinach was perfectly able to cope with the oxidative threatening due to low temperature as show by the AsA/DAsA ratio that was higher in low temperature than in the control leaves. This is particularly important for a leafy food vegetable like spinach, because increasing its nutritional quality by increasing the content of ascorbic acid would be unfeasible if linked to an increase of the nutritionally negative metabolite oxalic acid. The control of exchanges between two metabolite pools can be partially exerted by metabolic compartmentation. However, ascorbic acid is known to be present in many cellular compartments [18]. Scho¨ner and Krause [36] found that the rise of ascorbic

0

1,0 0,8 0,6 0,4 0,2 0,0

25 °C

10 °C

Fig. 3. Content of ascorbic in the lamina (Panel A), in the intercellular washing fluid (Panel B) and ascorbic acid/oxalic acid ratio in the intercellular washing fluid (Panel C) of spinach leaves grown at 25  C or acclimated to 10  C for 7 days. Data are the average  SE of three replicates.

acid under acclimation to low temperature, was not confined to chloroplasts, where most of the oxygen reactive species are produced, but was spread over other cellular compartments since its content in the bulk leaf lamina increased much more than in chloroplasts. Likewise, in both our experiments, the ascorbic acid content and the ascorbic/oxalic acid ratio increased in the apoplast after acclimation to 10  C. This shows that the burst of ascorbic acid

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caused by low temperature acclimation was not confined to specific cellular compartments. In particular it reached the apoplast, where the Green and Fry [14] ascorbic acid degradation pathway is located, making it unlikely that compartmentation would limit the ascorbic acid dependent synthesis of oxalic acid. Another site of control of the abundance of one metabolite in a specific tissue could be its export. It is unlikely that export from leaves is responsible for keeping the oxalic acid content low with respect to that of the ascorbic acid. Firstly because phloem export from leaves at low temperature is strongly reduced as shown by our carbohydrate data and as reported by Strand et al. [38] and secondly because the mobility of oxalic acid in leaves is limited to local diffusion, while ascorbic acid is loaded in phloem for long distance transport [10]. Degradation of one metabolite can indeed contribute to keep its concentration low in the presence of active synthesis. Oxalic acid can be degraded to CO2 and H2O2 by the enzyme oxalic acid oxidase. Oxalic acid degradation via OXO can then maintain a low content of oxalic acid even in the presence of an active synthesis. For example, rapid oxalic acid metabolism was found after feeding 14C oxalic acid to tobacco leaf discs [16]. OXO activity is present in several organisms ranging from bacteria [24] to fungi and higher plants [12]. In cereals, OXO activity is almost constitutively associated to germin proteins [5], frequently in the apoplastic cell compartment [8]. Our results indicate that it is unlikely that spinach leaf blade undergoes rapid oxalic acid degradation via OXO activity under normal temperature nor under 10  C acclimation. In conclusion, acclimation of spinach plants to growth at 10  C caused an increase of its nutritional quality that included a fully physiological increase of ascorbic acid that did not feed-forward on that of oxalic acid. Compartmentation of ascorbic acid in the plant cell, export to other tissues and degradation via OXO activity can all be reasonably discarded as causes of this lack of equilibrium between the two pools. Our results suggest, although direct and conclusive evidences should be obtained by further investigations, that the non-enzymatic conversion of ascorbic acid to oxalic acid is unlikely and that the ascorbic acid dependent synthesis of soluble oxalic seems to be under tight control, at least in spinach during acclimation to low growth temperature. This would give the possibility to breeders to increase the spinach content of ascorbic acid in order to improve its the nutritional quality. It also highlights the importance of environmental growing conditions in controlling the nutritional quality of plant foods. 4. Methods 4.1. Chemicals 4-aminoantipyrine, ascorbic acid, 2,2’dipyridyl, DL-dithiothreitol (DTT), ferric chloride (FeCl3), hydrogen peroxide (H2O2), N[2-Hydroxyethyl]piperazine-N0 -[2-ethanesulfonic acid] (Hepes), magnesium chloride (MgCl2), N,N’dimethylanilin, N-ethylmaleimide, oxalic acid, potassium chloride (KCl), potassium phosphate dibasic (K2HPO4), sodium hydroxide (NaOH), sodium acetate, sodium succinate, trichloroacetic acid (TCA), albumin from bovine serum (BSA), phosphoglucose isomerase, invertase were from Sigma (St. Louis, MO). Ethanol, amyloglucosidase, from Fluka (Sigma), ortho-phosphoric acid (H3PO4) were from Merck (Merck KGaA, Germany), hydrochloric acid (HCl) from Panreac (Barcelona Spain), potassium hydroxide (KOH) from BDH Chemicals (Ltd Poole England). Hexokinase, glucose phosphate dehydrogenase, a-amylase were from Roche (Mannheim Germany). Oxalic acid was measured by a test combination from r-biopharm (Roche, Mannheim Germany).

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4.2. Plant material and growth conditions Spinach (cv. Gigante invernale) were sown, and thinned to two plants, in 1 l plastic pots, containing a mixture of soil: sand: perlite (2:1:1, v/v). Plants were grown in growth cabinets (Fitotron SGD170 Sanyo Gallenkamp U.K.) under a 10 h light/14 h dark photoperiod. Photon flux density (PFD l 400–700 nm) was 800  5% mmol quanta m2 s1, while relative humidity was 70%  5% and CO2 concentration 360 ppm  10 ppm. Temperature was 25  0.5  C and 20  0.5  C for the light and dark period respectively. During the growth, plants were fed twice a week with a full nutrient solution and supplied with water as required. Acclimation to low temperature started 35 days after germination, by moving half of the pots to a twin cabinet, set with a day/night temperature regime of 10  0.5  C/10  0.5  C. 4.3. Sampling and analysis Measurements of gas exchange were made at PFD of 800 mmol quanta m2 s1 on the fourth fully expanded leaves, at the growing temperature, 4 and 28 h after the onset of acclimation, using a portable open gas exchange system (Li-6400, LI-COR, Lincoln, NE, USA). The specific leaf fresh weight (fresh weight/leaf area) and the specific leaf dry weight (dry weight/leaf area) were measured on leaf discs of 4 cm2 cut from the lamina with a sharp cork borer. Dry weight was obtained by freeze drying leaves to constant weight. All chemical analysis was performed on the fourth and fifth fully expanded leaves. 4.4. Leaf carbohydrate analysis Measurements of non-structural carbohydrate were performed on leaf discs, 1 cm2, collected from the leaf lamina with a cork borer and immediately frozen in liquid nitrogen. Frozen discs were ground 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 and extracted at 80  C for 45 min. The extract was centrifuged at 12 000 g for 5 min, soluble sugars (glucose, fructose and sucrose) were recovered in the supernatant, starch was in the pellet. Soluble sugars determination, by spectrophotometric coupled enzymatic assay, was performed as in Antognozzi et al. [2]. The carbohydrate assay 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 and 20 nkat of hexokinase. Shortly, the ethanol extract (10–20 ml in a total volume of 200 ml) was added to the assay solution, causing glucose and fructose to be phosphorylated to glucose-6 P and fructose-6 P by the hexokinase. After adding 5 nkat of glucose phosphate dehydrogenase, glucose is oxidised to 6-phospho gluconate with the stoichiometric production of NADH which is measured, after the reaction has reached the end point, as the change in absorbance at 340 nm (extinction coefficient ¼ 6.23 mM cm1). After reaching the end point for glucose measurement, 5 nkat of phosphoglucose isomerase were added, causing the isomerisation of fructose-6 P to glucose-6 P that eventually entered the previously described reaction causing a second rise of the NADH concentration. After the fructose reading, adding of 500 nkat of invertase, hydrolysed sucrose to glucose and fructose that were phosphorylated and entered the previously described reaction causing a third increase of NADH (two molecules of NADH produced for each sucrose molecule present in the extract). The pellet, containing starch, was washed three times with 50 mM NaAcetate buffer (pH 4.5) and then suspended and autoclaved at 120  C for 45 min in 1 ml of the same buffer. After autoclaving, 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 hydrolyse starch to glucose, that was then measured as described before. Chlorophylls were measured spectrophotometrically, using the ethanolic 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 liquid N2, and there stored until required. Discs were ground using a glass–glass homogenizer containing 1.5 ml of 10% (w/v) TCA, and centrifuged at 12 000 g for 15 min. AsA, and DAsA in the supernatant were measured using a colorimetric assay [20]. Total ascorbate (AsA þ DasA) was determined through a reduction of DAsA to AsA by 2 mM DTT. For AsA measurement, the assay solution (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 at 525 nm. For the total ascorbate (AsA þ DasA) measurement the assay 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 for 15 min. After reduction of DAsA to AsA the color was developed by adding 2.5% TCA, 0.8% 2,2’dipyridyl, 0.3% FeCl3 and the absorbance was measured at 525 nm. For each sample, DAsA was calculated as the difference between total ascorbate and reduced ascorbate concentrations. A standard curve in the range of 0–80 nmol ascorbate was used. 4.6. Determination of oxalic acid For the determination of oxalic acid leaf discs of 2.5 cm2, were extracted using a glass–glass homogenizer containing 1.5 ml of distilled water. The extract was centrifuged at 12 000 g for 5 min at 4  C. The supernatant was recovered for measurements of the soluble oxalic acid, the pellet was washed once with 1 ml of distilled water and re-suspended in 1.5 ml of water. After rapid adjusting of the pH to 2.8 with 1 N HCl, the suspension was placed at 50  C for 15 min for complete solubilization of oxalate salts, then the pH was adjusted to 5.0. Oxalic acid was measured using the enzymatic assay described by Beutler et al. [6]. Oxalate was cleaved to formic acid and CO2 at pH 5.0 in presence of oxalate decarboxylase. The formic acid formed was quantitatively oxidized to bicarbonate by NADþ at pH 7.5 in the presence of the enzyme formate dehydrogenase. The amount of NADH formed during the last reaction was measured as the 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 the intercellular washed fluid obtained as described by Pasqualini et al. [32]. Shortly, spinach leaf discs of 7 cm2 were washed with distilled water and vacuum infiltrated with a solution of KCl 100 mM pH 5.5 using 1 g of leaves in 100 ml of solution. The leaf material was then carefully placed in plastic syringes, centrifuged at 1500 g for 5 min a 4  C and the solution obtained was used for metabolite measurements. 4.8. Oxalate oxidase assay Oxalate oxidase (OXO; EC 1.2.3.4) cleaves one molecule of oxalic acid (þO2 and 2Hþ) to H2O2 and 2CO2. The OXO activity and was measured in the same leaves used for metabolites quantification, by the colorimetric assay described by Zhang et al. [39] with some modifications. Fresh leaves were ground in a glass–glass homogenizer containing distilled water (1:10 w/v) and after centrifugation at 5000 g for 8 min, the supernatant and pellet were checked for

OXO activity. The reaction solution (1 ml final volume), contained 50 mM sodium succinate pH 3.8, 5 mM oxalic acid, 20 ml/100 ml N,N’dimethylanilin, 8 mg/100 ml 4-aminoantipyrine, and 33.4 nkat ml1 of horseradish peroxydase. The reaction was started by adding the sample, and after incubation at 37  C for 20 min, it was stopped by adding 20 ml NaOH 1 M. The change in absorbance was measured at 550 nm. The H2O2 produced was estimated by comparison with a 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 Elisa plate reader (Anthos, 2001; Anthos Labtec Instruments, Salzburg, Austria) equipped with a kinetic software that allowed the record of the enzymatic reactions by reading absorbance changes every 15 s and that allowed the calculation of the absorbance difference between the start and the end of the reaction. Instrument internal error was 1 milli absorbance unit, assays were adjusted to have readings at least 100 times higher than the instrument’s internal error. 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 the whole procedure, was always higher than 90%. The recovery of soluble and insoluble oxalate was tested for the whole procedure [34] and it was always close to 100 with no effects of the procedure on the soluble/insoluble ratio. Chlorophyll, ascorbic acid and OXO activities were measured with a SIGMA ZWS11 spectrophotometer (ZWS 11 SIGMA BIOCHEM, Puchheim, Germany) in the single wavelength mode, the instrument has internal error lower than 1 milli absorbance unit, the assays were arranged in order to have readings at least 100 times higher than the instrument’s internal error. Recovery tests for ascorbic acid analysis gave recovery close to 95%. A recovery test of OXO activity in leaves was performed using root tissue that showed high OXO activity, the recovery was always close to 100%. Statistical analysis was performed by ANOVA using the STATISTICA software package (StatSoft for Windows, 1998). Variables were analysed by one way or two ways ANOVA as required by the experimental design, refer to table and figure legends for details. When the F test was significant, differences between averages were tested by an LSD with P ¼ 0.05. Acknowledgements The authors are indebted to Dr. Robert P. Walker for critical reading of the manuscript. This work was partially supported by the Italian 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 Tetragonia tetragonioides 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 of Actinidia 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 of flowering 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 Light and Carbohydrate Content on Acclimation of Spinach Photosynthesis to Low Temperature, Photosynthesis: from Light to Biosphere, vol. IV, Kluwer Academic 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 of Oxalate, Fresenius Zeitschrift fu¨r 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.

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