Beneficial effects of silicon on hydroponically grown corn salad (Valerianella locusta (L.) Laterr) plants

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Plant Physiology and Biochemistry 56 (2012) 14e23

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Research article

Beneficial effects of silicon on hydroponically grown corn salad (Valerianella locusta (L.) Laterr) plants Stefano Gottardi a,1, Francesco Iacuzzo a,1, Nicola Tomasi a, *, Giovanni Cortella b, Lara Manzocco c, Roberto Pinton a, Volker Römheld d, Tanja Mimmo e, Matteo Scampicchio e, Luisa Dalla Costa a, Stefano Cesco e a

Dip. Scienze Agrarie e Ambientali, University of Udine, 33100 Udine, Italy Dip. Ingegneria Elettrica, Gestionale e Meccanica, University of Udine, 33100 Udine, Italy Dip. Scienze degli Alimenti, University of Udine, 33100 Udine, Italy d Department of Plant Nutrition, University of Hohenheim, Stuttgart, Germany e Faculty of Science and Technology, Free University of Bolzano, 39100 Bolzano, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2012 Accepted 3 April 2012 Available online 20 April 2012

Soil-less cultivation of horticultural crops represents a fairly recent innovation to traditional agriculture which has several advantages including higher water-use efficiency. When plants are grown with this system, their roots come in contact with nutrients solely via the hydroponic solution. Although its beneficial effects have been widely demonstrated, silicon (Si) is mostly omitted from the composition of nutrient solutions. Therefore, the objective of this study was to assess the beneficial effect of Si addition to hydroponic solution on quali-quantitative aspects of edible production of two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown in soil-less floating system. Impacts on shelf life of this food were also studied. Results show that the supply of Si increased the edible yield and the quality level reducing the nitrate concentration in edible tissues. This result might be attributed to changes either in the metabolism (such as the nitrate assimilation process) or to the functionality of root mechanisms involved in the nutrient acquisition from the outer medium. In fact, our results show for the first time the ability of Si to modulate the root activity of nitrate and Fe uptake through, at least in part, a regulation of gene expression levels of the proteins involved in this phenomenon. In addition, the presence of Si decreased the levels of polyphenoloxidase gene expression at harvest and, in post-harvest, slowed down the chlorophyll degradation delaying leaf senescence and thus prolonging the shelf life of these edible tissues. In conclusion, data showed that the addition of Si to the nutrient solution can be a useful tool for improving quali-quantitatively the yield of baby leaf vegetable corn salad as well as its shelf life. Since the amelioration due to the Si has been achieved only with one cultivar, the recommendation of its inclusion in the nutrient solution does not exclude the identification of cultivars suitable for this cultivation system and the comprehension of agronomical and environmental factors which could limit the Si benefits. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Uptake Nitrate Iron Shelf life Hydroponic floating system

1. Introduction After oxygen, Silicon (Si) is the most abundant mineral element in soil [1] and comprises 28% of the earth’s crust [2]. However, Si is

* Corresponding author. Dipartimento di Scienze Agrarie e Ambientali, University of Udine, Via delle Scienze 208, I-33100 Udine, Italy. Tel.: þ390432558637; fax: þ390432558603. E-mail address: [email protected] (N. Tomasi). 1 Both authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2012.04.002

always combined with other elements, usually forming oxides or silicates and is rarely found in a free form. In the soil solution at pH values below 9, Si is present as silicic acid [Si(OH)4] at concentrations ranging from 0.1 to 0.6 mM, roughly two orders of magnitude higher than the concentration of phosphorous in the soil solution [1,3]. Plant roots absorb Si in the uncharged monomeric silicic acid form [4] and accumulate it up to 10% of the dry weight of their aboveground parts [5]. Among plant species, Si accumulation differs greatly, being the dicots, with respect to the monocots, unable to accumulate high levels of this element in their shoots [6,7]. Although Si accumulation levels are equivalent to those

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of macronutrient elements such as calcium, magnesium and phosphorous [1], Si has not been listed among the essential mineral elements for higher plants. However, the beneficial effect of this element has been observed in a wide variety of plant species [8], particularly when plants are under various stress conditions. In fact, the capability of Si to alleviate many abiotic and biotic stresses including physical stress (lodging, drought, radiation, high temperature, freezing, UV) and chemical stress (salinity, metal toxicity, nutrient imbalance) are widely described [1,4,6,8e12], even when Si has been applied at very low concentrations [13]. It is interesting to note that Si fertilization of soils characterized by low Si concentrations is common in order to increase the quantity and quality of several crops [14,15]. Conversely, due to its properties of un-dissociation at physiological pH and polymerization, high levels of Si in plant tissues are not toxic to the plant itself [16]. For these reasons, in all plants growing in the field where Si availability is not limited, Si taken up by roots might play an important role in alleviating an array of stresses to which these plants are constantly exposed. Si uptake mechanisms greatly depend on the plant species [17,18]. Si content of plants ranges from 0.1 to 10% (w/w) [1]. It has been hypothesized that there are three uptake/exclusion mechanisms: active, passive and rejection [19]. Recently many genes involved in Si transmembrane transport have been characterized in different plant species, demonstrating active root uptake transport from the rhizosphere [5,6,19,20] and xylem unloading [21]. However it has been demonstrated that passive transport of Si is present in at least some plant species [7,17,22]. Soil-less cultivation of horticultural crops represents a fairly recent innovation to traditional agriculture as it has several advantages including higher water-use efficiency, an aspect particularly important where water is becoming an economically scarce resource [23]. Among the various solutions, the floating system is one of the most suitable where a nutrient solution ensures the nutrient supply to plants. Therefore the levels of nutrients dissolved are often higher than those generally found in soil solution [24] and to guarantee the use of the hydroponic solution for more than one production cycle. The high NO 3 availability in the hydroponic solution [25] and sometimes even the unbalanced supply of some nutrients with respect to the others [26,27] may be a form of stress for the crops which in turn causes worsening of edible plant tissues such as nitrate accumulation, a feature particularly undesirable in leafy vegetables for its possible negative effects on human health [28]. On the other hand, it has been demonstrated that signs of leaf senescence, particularly undesirable for baby leaf yield, appear earlier and are more evident in plants grown with low nitrogen availability [29]. However, in addition to the composition of the nutrient solution, several other factors (such as the genetic make-up, the temperature, the light intensity, the photoperiod, etc.) can contribute to plant stress increasing the levels of nitrate accumulated in their tissues. It is interesting to note that for several of these stresses the beneficial effect of Si has been clearly demonstrated in alleviating their consequences [1,4,6,8,9,11]; even if the properties of Si are well documented, very frequently this element is omitted from the composition of the nutrient solutions for soil-less cultivation, particularly in those intended for the production of leafy vegetables. For these reasons, the objective of this study was to investigate the effect of Si on the yield of a leafy vegetable plant species grown in soil-less culture with a floating system. For this purpose, corn salad (Valerianella locusta (L.) Laterr.) plants have been grown in a nutrient solution with a composition widely adopted in greenhouse cultivation. At harvest, yield and quality have been evaluated in relation to the Si supply to the growth medium also considering aspects related to the shelf life of this product. Mechanisms of

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nitrate and Fe acquisition operating at the root level of plants supplied with Si, have also been investigated at the physiological and molecular levels. 2. Results Table 1 presents the values of marketable yield obtained by the two cultivars of Valerianella locusta (L. Laterr) after 45 days of cultivation in hydroponics with a floating system. Results showed that ‘Gala’ cultivar plants have significantly benefited from the addition of 30 mM Si in the growth medium increasing the yields by about þ61%. In these plants, the effect on leaf biomass accumulation exerted by Si is concomitant with an increase in the number of leaves per plant but did not show a significant difference in their leaf area (expressed in cm2 per g of leaf fresh weight); this aspect is also evident in the photo (Plate 1) of the leaf architecture which exhibited also a more erect position. In this cultivar, the effect of Si on yield is associated with an enhancement of SPAD index values and with increased water content in the leaves (estimated from the ratio dry/fresh leaf biomass weight). In contrast, plants of ‘Eurion’ cultivar, exhibited only a modest increase in biomass production of leaves (þ20%), with none of the other parameters being considerably affected by the exposure of their roots to Si in the nutrient solution. Results of nitrate analysis in the leaf tissues performed at harvest (Table 2) showed that in the cultivar ‘Gala’ the presence of Si decreased the nitrate concentration by approximately 17%. In contrast, no nitrate reduction could be observed for cultivar ‘Eurion’ plants when supplemented with Si. The treatment with Si did not change the content of S and P in leaves of either cultivar. Table 3 shows the contents of cationic nutrients determined in the leaf tissues by ICP-AES at the harvest. As expected, the addition of Si to the nutrient solution increased the concentration of the element at the leaf level, being significant only for the ‘Gala’ cultivar. In comparison to the control, the presence of Si in the growth medium caused a significant increase of Ca, Cu and Zn concentration in the leaves of cultivar ‘Gala’ plants, while the levels of K, Mg, Na, Fe and Table 1 Effect of Si on leaf yield and area of the two cultivars (‘Gala’ and ‘Eurion’) of corn salad (Valerianella locusta (L.) Laterr.) grown for 45 days in nutrient solution (NS) with a floating system in the absence (Control) or presence (þSi) of 30 mM Si. Data of leaf number, SPAD index and DW/FW ratio are also reported. Data are means  SD of three independent experiments; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05). Cultivar

Gala

Growth conditions

Control

þSi

Eurion

Leaf yielda (g FW m2) Leaf areab (cm2 g1) Number of leavesc (n per plant) SPAD indexd Ratio DW/FWe (%)

1313  91 C

2118  195 A 1421  129 C 1705  157 B

Control

þSi

36.5  3.6 A

34.6  4.1 A

40.6  3.2 A

39.5  6.0 A

6.5  0.9 B

8.3  0.6 A

5.3  1.2 B

6.3  0.7 B

33.0  1.2 B 35.9  0.8 A 32.1  0.9 B 33.4  0.8 B 8.28  0.19 A 6.88  0.17 B 8.39  0.04 A 8.55  0.40 A

a Two-factor ANOVA: FSi FSicv ¼ 9.282, P ¼ 0.016. b Two-factor ANOVA: FSi FSicv ¼ 0.0232, P ¼ 0.883. c Two-factor ANOVA: FSi FSicv ¼ 0.697, P ¼ 0.428. d Two-factor ANOVA: FSi FSicv ¼ 2.134, P ¼ 0.182. e Two-factor ANOVA: FSi FSicv ¼ 32.130, P < 0.001.

¼ 40.476, P < 0.001; Fcv ¼ 3.184, P ¼ 0.112; ¼ 0.363, P ¼ 0.564; Fcv ¼ 3.170, P ¼ 0.113; ¼ 8.055, P ¼ 0.022; Fcv ¼ 10.061, P ¼ 0.113; ¼ 14.617, P ¼ 0.005; Fcv ¼ 9.856, P ¼ 0.014; ¼ 20.323, P ¼ 0.002; Fcv ¼ 42.122, P < 0.001;

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Plate 1. Shoots of the two cultivars ‘Gala’ and ‘Eurion’ of corn salad (Valerianella locusta (L.) Laterr.) grown for 45 days in nutrient solution (NS) with floating system in the absence (Control) or presence (þSi) of 30 mM Si. Scale bar ¼ 15 mm.

Mn accumulated in the leaf of these latter plants were unaffected by Si exposition to their roots. In contrast, in plants of cultivar ‘Eurion’, the treatment with Si did neither affect the nutrient contents (Ca, K, Na, Mn, Zn) of leaves nor significantly lowered some of them (Mg, Cu and Fe). When Si was included in the composition of the nutrient solution, in plants of ‘Gala’ cultivar a significant increase in the dry matter accumulation of roots was observed (Table 4). The application to the root pictures of the ImageJ program made it possible to calculate the length and the area of these tissues which showed a two fold increase in Si-fed plants of ‘Gala’ cultivar compared to those of the control. Differently, when plants of ‘Eurion’ cultivar are considered, the presence of Si in the growth medium did not affect either their biomass accumulation at the root level, the length or the surface area of these tissues. In order to evaluate whether the supplementation of Si to the nutrient solution could create conditions at the leaf level which could contribute to prolonging the shelf life of the marketable yield, the transcript levels of a gene coding for a polyphenoloxidase involved in post-harvest oxidation of edible leaf material, have

been evaluated at harvest. Results reported in Fig. 1 showed that in cultivar ‘Gala’ the exposure of roots to 30 mM Si reduced the expression level of this gene. Evaluation of the expression levels of NR (coding for a nitrate reductase which is involved in the first step of the nitrogen assimilation pathway) gene were also performed showing a significant up-regulation of this gene but only in ‘Gala’ cultivar when grown in the presence of Si (Fig. 1). The quality evolution of these edible tissues during their postharvest storage at 4  C was evaluated measuring SPAD index values, fresh weight and nitrate contents in leaves. As reported in Fig. 2 and Suppl. Table 1, in both cultivars the values of SPAD index decreased progressively with the storage time regardless of treatments, signaling indirectly a loss of greenness of the leaves [30], and therefore a decrease in the chlorophyll concentration. When plants were grown in the nutrient solution containing Si the rate of this leaf decolourization was significantly decreased, up to 36% in the case of the cultivar ‘Gala’. Due to the importance of salad color on its overall sensory acceptability, the shelf life of corn salad at 4  C was assessed on the basis of the changes in the SPAD index value [31] for this purpose, the acceptability limit [32] was chosen

Fig. 1. Real-time RT-PCR analyses of polyphenoloxidase (PPO) and nitrate reductase (NR) gene expressions in leaves of the two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Gene mRNA levels were normalized with respect to the internal control alpha-tubulin; relative changes in gene expression were calculated on the basis of their expression levels in control plants. Bars represent means  SD of transcript levels on 2 independent experiments with 3 replicates; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05).

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Fig. 2. Effect of Si on SPAD index values of corn salad leaves during post-harvest storage at 4  C. Data are means  SD of seven independent experiments, table resuming the results of the Three-factor ANOVA, Fischer LSD is presented as supplementary data (Suppl. Table 1).

corresponding to the SPAD index value associated to the minimum color change potentially perceivable by the consumers. While no effect of Si was detected on the shelf life of corn salad of the ‘Eurion’ cultivar, it significantly affected product stability in the case of the ‘Gala’ one. In particular, the presence of Si in the nutrient solution extended shelf life by 100% (Table 5). The fresh weight values of these leaf tissues were essentially constant throughout the storage period for the ‘Gala’ cultivar while in the ‘Eurion’ cultivar they showed a gradual decline (Fig. 3A). These changes were significantly influenced by the presence or absence of Si in the growth medium for either cultivar (Suppl. Table 2). Analysis of the nitrate contents in the leaf tissues highlighted in ‘Eurion’ cultivar a marked decrease (about 60%) in the anion concentration during storage, regardless of the hydroponic integration with Si (Fig. 3B, Suppl. Table 3). In contrast, no changes during the storage was observed in the anion content of leaves collected from ‘Gala’ cultivar plants; however, when Si was added to the nutrient solution, in leaves of this latter cultivar a slow and gradual decline in the nitrate contents was observed. In order to evaluate the functionality of acquisition mechanisms of nutrients whose availability in the growth medium has been demonstrated to be linked with the nitrate accumulation at the leaf level [25,27], nitrate and iron uptake for both cultivars were

measured using roots excised from plants at the harvest stage. Results reported in Fig. 4 show that plants of ‘Gala’ cultivar, when grown in the presence of Si, induced a rise in the capacity to take up nitrate (þ67%); conversely, the treatment with Si of cultivar ‘Eurion’ plants did not affect the rate of nitrate uptake. Fig. 5 A shows that the presence of Si in the growth medium favored the development of higher uptake rates of 59Fe in both cultivars. Since corn salad plants, like other dicots, acquire Fe from the external medium via a mechanism, called Strategy I [33], which involves an obligatory reduction of ferric ion [34] prior to membrane influx of Fe2þ [35], Fe(III)-chelate reductase activity has been also assayed. Results reported in Fig. 5 B show that the higher 59Fe uptake observed in Sifed plants was also associated with higher levels of Fe(III)-chelate reducing activity. In contrast, the proton extrusion capacity of roots (calculated on the basis of the pH changes of the nutrient solution which were measured weekly), showed a significant decrease from the presence of Si in the growth medium (Fig. 5 C) but only in ‘Gala’ cultivar where, on the other hand, an enhanced nitrate uptake via proton co-transport [36] took place. In order to evaluate a transcriptional regulation of membrane proteins involved in the acquisition of these two nutrients, the transcript abundance of LATS (coding for a low affinity nitrate transporter) and FRO (coding for an isoform of the PM Fe(III)-

Fig. 3. Effect of Si on leaf biomass of corn salad during post-harvest storage at 4  C (A). Values of nitrate content in the leaves are also reported (B). Data are means  SD of three independent experiments, tables resuming the results of the Three-factor ANOVA, Fischer LSD is presented as supplementary data (Suppl. Tables 2 and 3).

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the addition of Si to the nutrient solution, in roots of cultivar ‘Gala’ a significant down-regulation of this gene was evident as a consequence of nutrient solution amendment with Si. 3. Discussion

Fig. 4. Effect of Si on NO 3 uptake by roots of the two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Net nitrate uptake was measured spectrophotometrically as depletion from a solution containing 1 mM NO 3. Data are means  SD of three independent experiments; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05).

chelate reductase) genes were analyzed in roots of corn salad grown in the presence or absence of Si. Results reported in Fig. 6 showed that in ‘Gala’ cultivar, Si-fed plants exhibited a significant increase in the expression levels of LATS and FRO genes. Differently, in roots of ‘Eurion’ plants only the transcript abundance of FRO gene was significantly enhanced by the exposure to Si. In Fig. 6, transcript levels of a gene coding for an isoform of the silicon transporter (LSI), are also reported. Results show that, while in ‘Eurion’ cultivar roots the expression level of this gene was unaffected by

When plants of crop species such as corn salad (Valerianella locusta (L.) Laterr.) are grown in soil-less cultivation systems, their roots come in contact with nutrients solely via the hydroponic solution. Although its beneficial effects have been widely demonstrated for monocots and dicots (for review see Refs. [5] and [37]), Si is mostly omitted from the composition of commercial nutrient solutions. For this reason, the objective of this study was to evaluate whether the addition of Si to the hydroponic solution had any beneficial effects in the quali-quantitative aspects of edible production of corn salad grown in soil-less system. Impacts on shelf life of this food were also studied. Results show that the supply of 30 mM Si via the nutrient solution to hydroponically grown corn salad made it possible to increase the edible yield of ‘Gala’ cultivar (þ61%) and ‘Eurion’ cultivar (þ20%) (Table 1). This result is in agreement with studies on other crop species showing the ability of Si to stimulate the growth and development of plants, particularly under abiotic and biotic stresses [1,5,8], even when applied at very low levels [13]. In our conditions, when ‘Gala’ cultivar is considered, the yield increase promoted by the presence of Si in the nutrient solution was the result of an enhancement of both the weight of each leaf and the leaf number per plants (Table 1) which exhibited also a more erect position (Plate 1). It is interesting to note that this beneficial effect of Si has been attributed, at least in part, to the anatomical changes imposed by silica deposition in cell walls which in turn keeps leaves erect and improves light interception by these tissues thereby stimulating photosynthesis as described for rice [4]. Although it is well known that graminaceous species accumulate high levels of Si, in hydroponic systems where roots have very low access to silicon, even plants with low Si needs, like dicots, might suffer of Si deficiency. Moreover in Si-fed plants, the higher levels of chlorophyll (Table 1) could further contribute to the phenomenon, as also observed by Liang [38] in barley plants. Ma and Yamaji [6] argued that leaf position is particularly important to minimize mutual leaf

Fig. 5. Effect of Si on 59Fe uptake (A) By roots of the two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Iron uptake was determined at pH 6.0 by using 59Fe(Fe-EDDHA, final Fe concentration of 100 mM) tracers. Root Fe(III)-chelate reducing activity (B) and Hþ extrusion into the nutrient solution (C) By roots of the two cultivars, are also reported. Data are means  SD of three independent experiments; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05).

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Fig. 6. Realtime RT-PCR analyses of LATS (coding for a low affinity NO 3 transporter), FRO (coding for an isoform of the PM Fe(III)-chelate reductase) and LSI (coding for an isoform of the silicon transporter) gene expression in roots of the two cultivars (‘Gala’ and ‘Eurion’) of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Gene mRNA levels were normalized with respect to the housekeeping gene alpha-tubulin; relative changes in gene expression were calculated on the basis of expression levels of alpha-tubulin in roots of control plants. Bars represent means  SD of transcript levels on 2 independent experiments with 3 replicates; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05).

shading in dense plant stands and when nitrogen fertilizers are heavily applied, which are growth conditions very similar to those generally used for the production of baby leaf yield in soil-less systems. With respect to light, its intensity has been identified as one of the major factors causing, when not appropriate to the plant needs, high levels of nitrate accumulation in leafy vegetable tissues [28], thus worsening their quality for consumption of babies. In our work, the analysis of nitrate contents in the leaves of ‘Gala’ cultivar

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showed that Si, concomitantly to the stimulation of plant growth, limited the nitrate accumulation in the edible tissues (Table 2). These effects could only partially be related to anion dilution as a consequence of the higher foliar biomass and its water content which we found in plants treated with Si (Table 1). In fact, it is interesting to note that the lowering in leaf nitrate content was in parallel to an increase in the root’s capacity to take up nitrate from the outer solution (Fig. 4) suggesting a nitrogen assimilation process more pronounced in Si-fed plants of this cultivar. The higher levels of transcripts of the gene coding for a nitrate reductase (Fig. 1) at the leaf level, support this idea. On the other hand, evidence of changes in plant metabolism related to the presence of Si in the growth medium is well described for plant resistance to insect attack which involves the production and the release of volatile phenolic compounds with anti-nutritional, deterrent and toxic properties (see the reviews [37]). It has been also reported that Si-treated maize plants release fifteen times more phenolics than those untreated [39]; the role of these compounds in the nutrient availability is widely accepted (see reviews [40] and [41]). Furthermore, Liang et al. [5] support the idea that Si may even be actively involved in metabolic and/or physiological activities of plants. When Si has been included in the composition of the nutrient solution, the ability of plants to accumulate a greater level of leaf biomass requires an enhanced nutrient acquisition by roots from the external medium in addition to higher levels of photosynthetic activity. As previously stated, in ‘Gala’ cultivar the presence of Si in the hydroponic solution enhanced root capability to take up nitrate from the external solution (Fig. 4) involving in this phenomenon a regulation at the gene expression level (Fig. 6) of a low affinity transport system (LATS [42]). The greater capacity to acquire nitrate from the external medium requires higher levels of protons for the transmembrane anion co-transport [36] thus leading to rises in the pH values of the growth medium (Fig. 5). In this cultivar, even the root acquisition of Fe from the outer medium increased in Si-fed plants (Fig. 5) and this aspect is particularly important for the role exerted by this micronutrient in the levels of nitrate accumulation in plant tissues. In fact, it is well demonstrated that high levels of nitrate are accumulated at the leaf level of Fe-deficient plants [26,27] which could represent a problem for corn salad yield causing a worsening of its quality [28]. Dicots, such as corn salad plants, acquire Fe from the external medium via a mechanism [33] which involves an obligatory reduction step of ferric ion prior to its membrane influx. Our results show that the higher acquisition of 59Fe by Si-treated plants is guaranteed by the enhanced activity (Fig. 5) and transcript abundance (Fig. 6) of Fe(III)-chelate reductase. However, despite the greater root capacity to acquire Fe when plants are grown in the presence of Si, the micronutrient content of their leaves has not been increased (Table 3). The need of Si-fed plants to meet the greater demands for Fe due to increased plant growth without rising Fe contents that are toxic for the cells, could explain this pattern observed in ‘Gala’ cultivar. Also in plants of ‘Eurion’ cultivar the addition of Si to the nutrient solution increases the capability of their roots to acquire Fe from the external medium; however, the lack of any significant benefits in yield for the plants by Si, suggests that some other agronomic or environmental factor could play a more crucial role in the qualiquantitative aspects of the edible yield of this cultivar. The calculation of dry/fresh weight ratio of the leaf biomass highlighted (Table 1) higher water contents in Si-fed plants of ‘Gala’ cultivar at the harvested stage. In this regard, it has been argued by Romero-Aranda et al. [43] that the hydrophilic nature of silicon could help to keep water within the leaf tissues where Si can also form a barrier that reduces water loss through the cuticle via crystals deposited in epidermal cells [44]. The enhanced leaf

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Table 2 Concentrations of NO 3 , S and P in leaf samples of the two cultivars (‘Gala’ and ‘Eurion’) of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Data are means  SD of three independent experiments; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05). Cultivar

Gala

Growth conditions Control

Eurion þSi

þSi

Control

a (g kg1 NO 3.82  0.15 A 3.18  0.23 B 4.17  0.29 A 3.90  0.27 A 3 leaf FW) b 1 S (g kg leaf FW) 0.63  0.05 B 0.65  0.10 B 0.94  0.12 A 1.12  0.19 A Pc (g kg1 leaf FW) 1.38  0.09 A 1.26  0.06 A 1.07  0.07 B 1.05  0.15 B a Two-factor ANOVA: FSi ¼ 10.801, P ¼ 0.011; Fcv ¼ 14.732, P ¼ 0.005; FSicv ¼ 1.805, P ¼ 0.216. b Two-factor ANOVA: FSi ¼ 1.846, P ¼ 0.211; Fcv ¼ 29.541, P < 0.001; FSicv ¼ 1.372, P ¼ 0.275. c Two-factor ANOVA: FSi ¼ 1.345, P ¼ 0.280; Fcv ¼ 20.450, P ¼ 0.002; FSicv ¼ 0.756, P ¼ 0.410.

hydration could contribute, at least in part, to delaying leaf senescence and to slowing down chlorophyll degradation (Fig. 2) thus prolonging the shelf life of these edible tissues (Table 5). The capability of Si to decrease the permeability of plasma membranes and the membrane lipid peroxidation [12,45], as well as affecting the activity of polyphenoloxidase enzyme via regulation of its gene expression in the edible leaf material (Fig. 1) would also encourage this. In common with that described for salinised plants [43], the higher water content of the leaves in Si-fed plants coupled with their enhanced biomass could also contribute to salt dilution allowing greater nutrient accumulation such as those measured for Ca, Cu and Zn in the leaf tissues of ‘Gala’ cultivar (Table 3).

Table 3 Cationic nutrients in leaf samples of the two cultivars (‘Gala’ and ‘Eurion’) of corn salad (Valerianella locusta (L.) Laterr.) grown as described in Table 1. Data are means  SD of three independent experiments; capital letters refer to statistically significant differences among the samples (Two-factor ANOVA, Fischer LSD, d.f. ¼ 1, P < 0.05). Cultivar

Gala

Growth conditions Control Caa Kb Mgc Nad Cue Fef Mng Znh Sii

Eurion +Si

g kg1 leaf DW 1.81  0.10 B 3.08 17.1  1.3 A 15.1 0.70  0.05 B 0.81 0.09  0.02 B 0.09 mg kg1 leaf DW 0.81  0.09 B 1.19 82.4  4.2 AB 79.0 55.4  2.9 B 57.4 18.3  1.9 B 29.0 1.43  0.31 B 3.17

Control

+Si

   

0.21 A 1.3 A 0.04 B 0.01 B

2.17 16.6 1.05 0.05

   

0.25 B 1.9 A 0.11 A 0.01 A

1.87 16.9 0.79 0.04

   

0.19 B 1.2 A 0.05 B 0.01 A

    

0.11 A 7.1 AB 6.2 B 2.1 A 0.25 A

1.11 88.0 69.8 19.1 1.61

    

0.17 A 6.1 A 5.1 A 1.6 B 0.16 B

0.86 72.6 65.4 19.8 1.79

    

0.14 B 7.1 B 3.4 AB 1.8 B 0.07 B

a Two-factor ANOVA: FSi ¼ 18.086, P ¼ 0.003; Fcv ¼ 13.874, P ¼ 0.006; FSicv ¼ 47.505, P < 0.001. b Two-factor ANOVA: FSi ¼ 1.035, P ¼ 0.339; Fcv ¼ 0.612, P ¼ 0.457; FSicv ¼ 1.893, P ¼ 0.206. c Two-factor ANOVA: FSi ¼ 4.054, P ¼ 0.079; Fcv ¼ 17.655, P ¼ 0.003; FSicv ¼ 24.031, P ¼ 0.001. d Two-factor ANOVA: FSi ¼ 0.222, P ¼ 0.650; Fcv ¼ 32.000, P < 0.001; FSicv ¼ 0.222, P ¼ 0.650. e Two-factor ANOVA: FSi ¼ 0.705, P ¼ 0.426; Fcv ¼ 0.0417, P ¼ 0.843; FSicv ¼ 18.384, P ¼ 0.003. f Two-factor ANOVA: FSi ¼ 6.816, P ¼ 0.031; Fcv ¼ 0.0123, P ¼ 0.914; FSicv ¼ 2.775, P ¼ 0.134. g Two-factor ANOVA: FSi ¼ 0.204, P ¼ 0.663; Fcv ¼ 17.814, P ¼ 0.003; FSicv ¼ 1.451, P ¼ 0.263. h Two-factor ANOVA: FSi ¼ 28.668, P < 0.001; Fcv ¼ 15.640, P ¼ 0.004; FSicv ¼ 21.248, P ¼ 0.002. i Two-factor ANOVA: FSi ¼ 44.821, P < 0.001; Fcv ¼ 17.477, P ¼ 0.003; FSicv ¼ 29.568, P < 0.001.

3.1. Conclusions In conclusion, data here presented showed that the supplementation of Si to the nutrient solution used in soil-less culture can be a useful tool for improving quali-quantitatively the yield of baby leaf vegetable corn salad and its shelf life. Our results indicate that the benefits of this addition are improvements on both the levels of productivity and quality (i.e. the reduced nitrate levels in edible tissues). This result has been a consequence of changes either in the metabolism (such as the nitrate assimilation process) or in the functionality of root mechanisms involved in the nutrient acquisition from the outer medium. In fact, our results showed for the first time the ability of Si to modulate the root activity for nitrate and Fe uptake through, at least in part, a regulation of gene expression levels of the proteins involved in this phenomenon. Since the amelioration due to Si have been achieved only with the cultivar ‘Gala’, the recommendation of its inclusion in the nutrient solution [46] does not exclude the identification of cultivars suitable for this cultivation system and the comprehension of agronomical and environmental factors which could limit the Si benefits in cultivars like ‘Eurion’. 4. Methods 4.1. Plant material and growth conditions Plants were grown hydroponically in a floating system composed of expanded polystyrene boards floating on a nutrient solution contained in rectangular pots (approximately 0.1 m2 surface, 20 dm3 capacity) with the same ratio between nutrient-solution volume and plant number as in greenhouse cultivation. The trials were carried out in a growth chamber as described by Cesco et al. [47] with the following controlled climatic conditions: day/night photoperiod, 16/8; radiation, 220 mE m2 s1; temperature (day/ night) 25/20  C; RH 70e80%. Corn salad seeds (Valerianella locusta (L.) Laterr.; cultivar ‘Gala’ and cultivar ‘Eurion’ from DOTTO SpA, Italy), were manually sown upon boards (on holes arranged in rows at a distance of approximately 3 cm apart) with perlite (BPB e Italia SPA, Milano, Italy).Then, the boards were placed on rectangular pots containing 1 mM CaSO4 in darkness for 5 days at 27  C and 90% relative humidity for seed germination. After emergence, in order to obtain a density of 1800 plants m2, seedlings were counted and the excess removed. Then, the boards were transferred into rectangular pots containing the following aerated nutrient solution slightly þ þ modified from Zanin et al. [48]: (mM) NO 3 15, NH4 3, P 3.5, K 11, 2þ 2þ 2þ 2þ Ca 4.5, Mg 3.5, S 6; (mM) Mn 10, Zn 5, H3BO3 40, Cu2þ 1, 3þ MoO2 40 (pH adjusted at 6.0 with 1 M KOH). In order to 4 0.5, Fe ensure the availability of the Fe chelate, the highly stable complex Fe(III)-EDDHA [49] has been used; the complex was prepared according to Bar-Ness et al. [50] by mixing FeCl3 with o,o-EDDHA with a molar ratio of 1:1.1. The solution was aerated by bubbling which also guaranteed the constant mixing of the solution. In order to evaluate the effect of Si availability on the corn salad yield and quality, the nutrient solution was supplemented with 30 mM Si (applied as Na2SiO3). This treatment was carried out for half of the pots. As control, the remaining half of the pots was maintained with the nutrient solution in the original composition without any supplementation. Distilled water used for the preparation of nutrient solution had a Si concentration