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Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency
Accepted Manuscript Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency a a M Blanca Pascual, Virginia Echevarria, M José Gonzalo, Lourdes HernándezApaolaza PII:
S0981-9428(16)30275-3
DOI:
10.1016/j.plaphy.2016.07.008
Reference:
PLAPHY 4616
To appear in:
Plant Physiology and Biochemistry
Received Date: 19 April 2016 Revised Date:
6 July 2016
Accepted Date: 9 July 2016
Please cite this article as: M.B. Pascual, V. Echevarria, M.J. Gonzalo, L. Hernández-Apaolaza, Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency, Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.07.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency
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Mª BLANCA PASCUAL, VIRGINIA ECHEVARRIA, Mª JOSÉ GONZALO, LOURDES HERNÁNDEZ-APAOLAZA Agricultural Chemistry and Bromatology Department, Francisco Tomas y Valiente Street, n 7. Autonoma University 28049, Madrid, Spain [email protected] [email protected] [email protected]
Abstract
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Corresponding Author: Lourdes Hernández-Apaolaza [email protected] Phone: +34 914976859 Fax: +34 914973825
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[email protected]
It is well established the beneficial role of silicon (Si) in alleviating abiotic stress. However, it remains poorly understood the mechanisms of the Si-mediated
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protection against metal deficiency, especially the zinc (Zn) one. Recently, it has been proposed that Si may act by an interaction with this biometal in the root apoplast contributing to its movement through the plant, as in the case of Fe deficiency. In the present work, the effect of initial or continuous Si doses in
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soybean Zn deficient plants has been studied. For that purpose, plants grown in hydroponic culture were treated with different Si doses (0.0, 0.5 and 1.0 mM)
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under Zn limiting conditions. SPAD index in leaves, several growth parameters, mineral content in the whole plant and the formation of Zn pools in roots were determined. An initial addition of 0.5 mM of Si to the nutrient solution led to an enhancement of plants growth, Zn and Si content in leaves, and a higher storage of Zn in the root apoplast. The results suggest that this treatment enhanced Zn accumulation on roots and its movement to shoots when needed, mitigating Zn deficiency symptoms.
Key words: apoplast; silicon; soybean; zinc deficiency; Zn-Si interaction.
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1. Introduction Zinc (Zn) deficiency cause important disturbances in growth and development of in plants due to the large diversity of essential cellular functions and
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metabolic pathways directly influenced (Cakmak, 2000). Severe symptoms such as intervenial chlorosis in leaves, reddish-brown or bronze tints, epinasty, internode shortening, inward curling of leaf lamina and reductions in leaf size have been associated to Zn deficiency (Marschner, 1995).
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Although silicon (Si) is the second most abundant element in the earth’s crust, it is not still considered an essential element for higher plants. However, it has
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been largely known its beneficial effects alleviating various biotic (diseases, pests) and abiotic (salt and metal toxicity, high temperature, drought, freezing) stresses in many plant species (Epstein, 1999; Maksimovic et al., 2012; Romero et al., 2011; Song et al., 2011; Wu et al., 2013; Zhu and Gong, 2014). Moreover, in the past years there has been a rapid progress in the elucidation of how Si mediates under plant metal deficiency (Bityutskii et al. 2014; Gonzalo
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et al., 2013; Hernández-Apaolaza, 2014; Mehrabanjoubani et al., 2015; Pavlovic et al., 2013). However, despite the information available, the mechanisms of Si-mediated alleviation of nutrient deficiency in crops remain poorly understood.
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There are several evidences that Zn distribution in plant changed by the Si addition in hydroponic and soil experiments (Bityutskii et al., 2014; Gu et al.
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2012) and that both elements presented a similar location in plants (Gu et al., 2012). By using a fractionation technique, it has been shown that the cell wall bound fraction of Zn in roots, stems, sheaths and leaves of rice seedlings increased after Si addition (Gu et al., 2012). Moreover, in Fe plaque, Zn could be adsorbed (Chen et al. 1980) and remobilized when needed. Root Zn deposits could be used under Zn-deficient conditions through the activation of a Zn-deficiency mechanism (Hernández-Apaolaza, 2014). As for Fe (Pavlovic et al., 2013), the Zn apoplastic pools in the roots could be more mobile under deficiency conditions when Si was added, contributing to its better distribution inside plant (Hernández-Apaolaza, 2014). Recently, Bityutskii et al., (2014)
ACCEPTED MANUSCRIPT studied the effect of Si on Zn deficiency in acidic condition (nutrient solution pH 6.0) by growing cucumber seedlings without Zn and two Si doses (0 and 1.5 mM) in hydroponic culture. Even though no significant changes were observed in roots or leaves’ Zn and chlorophyll concentrations due to Si addition, Si partially diminished leaves necrotic spots. The reutilization of root apoplastic Fe
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via phenolics induced by Si nutrition did not appear under Zn deficiency and authors concluded that there were no evidences of Si alleviation of Zndeficiency symptoms, but as plants were grown without Zn in the nutrient solution, the possibility of Zn pools formation was suppressed, therefore their
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remobilization was not possible.
The aim of this work was to investigate the effect of Si addition on Zn deficient symptoms mitigation and the distribution of both elements, Zn and Si, in
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soybean seedlings under Zn limited conditions. In order to accomplish this objective different Si doses were applied to soybean plants grown in a nutrient solution with a deficient Zn content. The Zn deposits in apoplast were determined and its remobilization from root to shoot was also investigated.
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2. Materials and methods
2.1. Plant material and growing conditions Soybean seeds (Glycine max L. cv. Klaxon) were germinated on filter paper moistened with distilled water for one week in the dark at 28ºC. Thereafter,
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uniformly sized seeds were grown in aerated 1/5 strength nutrient solution for a week. Then, they were transferred to 2 L plastic buckets containing full-strength
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nutrient solution in which 0.2 g of solid CaCO3 were added per bucket. The composition of the nutrient solution was: macronutrients (mM) 1.0 Ca(NO3)2, 0.9 KNO3, 0.3 MgSO4, and 0.1 KH2PO4; micronutrient (µM) solution to avoid metal precipitations 35 NaCl, 10 H3BO3, 0.05 Na2MoO4, 50 Fe (III)-HBED, 10 ZnEDTA, 2.5 MnSO4, 1.0 CuSO4, 1.0 CoSO4, 1.0 NiCl2, and 115.5 EDTANa2 and 0.1 mM HEPES, 0.05 mM KOH. The solution was continuously aerated and renewed weekly. The pH was adjusted to 7.5 with NaOH 1.0 M or HCl 1.0 M. When necessary, the buckets were refilled with distilled water. Soybean plants were submitted to different Si concentrations in presence/absence of Zn depending on the experiments.
ACCEPTED MANUSCRIPT For the preparation of the Fe chelate solution, HBED (N,N'-bis(2-hydroxybencyl) ethilendiamine-N,N'diacetic acid, Strem Chemicals) was dissolved in sufficient NaOH (1/3 molar ratio) and then complexed with FeCl3·6H2O (Merck). After adjusting to 7.0 its pH, the solution was left to stand overnight to allow excess Fe to precipitate. Finally, the solution was filtered through 0.45 µm Millipore
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membrane and made up to volume. The Zn was supplied as Zn-EDTA, which was prepared by mixing at 1/1 molar ratio Zn(NO3)2·6H2O (98%. Sigma-Aldrich) and ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich).
Plants were grown under controlled conditions in a Dycometal-type CCK growth chamber provided with fluorescent and sodium vapor lamps with a 350 µE m−2
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s−1 light intensity, photoperiod 16/8 h, temperature (day/night) 30/20°C and
2.2. Experimental design.
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50/70% relative humidity.
2.2.1. Effect of Si in the formation and remobilization of the apoplast Zn pool Soybean plants, grown as mentioned above, were cultivated with a full strength nutrient solution for two weeks. Three different Si doses were added: 0.0, 0.5 and 1.0 mM as Na2SiO3·5H2O (Suprapur, Merk). Then, Zn supply was
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eliminated from all the nutrient solutions except for the plants of the control treatment and the Si was maintained in half of the plants (continuous silicon supply) for four more days. Table 1 gathers the six different treatments that
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were carried out.
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Table 1: Silicon and Zinc treatments (mM and µM, respectively) regarding to the days after treatments application (DAT).
Si (mM)
Zn (µM)
0-14 DAT
14-18 DAT
0-14 DAT
14-18 DAT
Abbreviation
0.0
0.0
0
0
Zn 0 Si (0.0-0.0)
0.0
0.0
10
10
Zn10 Si(0.0-0.0)
0.5
0.0
10
0
Zn 0 Si(0.5-0.0)
0.5
0.5
10
0
Zn 0 Si(0.5-0.5)
1.0
0.0
10
0
Zn 0 Si(1.0-0.0)
1.0
1.0
10
0
Zn 0 Si(1.0-1.0)
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hydroponic experiment lasted a total of five weeks.
2.3. Measurements.
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weeks after the Zn removal from the nutrient solution. Therefore, this
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In the first experiment described, the root apoplastic accumulation of Zn was tested after Zn removal from the nutrient solution. The method used was the one proposed by Rengel (1999) in which intact roots were washed twice in a
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mixture of distilled water and ice for 5 minutes each, and then with a solution containing 2 mM CaCl2 and 1 mM LaCl3 for 10 minutes in continue stirring. This last washed solution was filtrated, made up to 50 ml and Zn concentration was assessed by atomic absorption spectrophotometer (AAs, Perkin-Elmer Analyst 800). In addition, the washed roots were oven dried, and Zn content was measured after a microwave (CEM Corporation MARS 240/50; Matthews, NC,
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USA) digestion following the method described by Zuluaga et al. (2011) to determine the total amount of Zn in roots and its distribution. For that, 500 mg of dry material were loaded into Teflon vessels with 8 mL HNO3 at 65%, 2 mL
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H2O2 at 30% and 1 mL HF at 40% (all of them Suprapure reagent grade, Merck Millipore). After the digestion the samples were made up to 50 mL volume with distilled water and Zn concentration was determined by AAs.
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During the whole long term experiment, the SPAD index was monitored every three days using a digital chlorophyll meter SPAD (Soil and Plant Analyzer Development) model 502 (Minolta Co., Osaka, Japan). Samples of leaves, stems and roots of three plants per treatment were taken at 0(M0), 7 (M1), 14(M2) and 21 (M3) days before the Zn removal of the nutrition solution and fresh weight (FW) was determined. Immediately after the sampling, plants were washed with four different solutions: Tween 80 at 0.1%, 0.1 M of HCl (Suprapure, Merck Millipore), water and distilled water. Then, the biometric parameters dry weight (DW), stems length (SL), roots length (RL), and node
ACCEPTED MANUSCRIPT number (NN) were measured. In addition, a sample of all the nutrient solutions was taken before every change for their pH measurement and nutrient analysis. Fe, Cu, Mn, and Zn concentrations were determined in leaves, stems and roots following the previously described microwave method. Silicon was determined by digesting 20 mg of ground plant materials in 1.0 ml of
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the mixture of 1 M HCl and 2.3 M HF (v/v = 1/2) and shaken overnight. The samples were centrifuged at 10 g for 10 minutes, and 0.5 ml of the supernatant was added to plastic tubes. Afterwards, 3.0 ml of 20% glacial acetic acid and 1.0 ml of 54 g.L-1 (NH4)6Mo7O24·4H2O were added and incubated for 5 minutes,
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followed by addition of 0.5 ml of 20 g.L-1 tartaric acid and 2.0 ml reductant. Thirty minutes after the reductant addition, the sample absorbance was measured at 650 nm with a spectrophotometer (Jasco V-650). The reductant
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was a mixture of solution A: 80 g.L-1 Na2SO3 + 16 g.L-1 1-amino-2-naphthol-4sulfoacid and solution B: 12.5 g.L-1 NaHSO3. Then, the solution A and B were mixed together and made up to 250 ml (Iwasaki et al., 2002; Novozamsky et al., 1984, Van der Vorm, 1987)
2.4. Statistical analysis
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Obtained data were analyzed by using the IBM SPSS Statistics Version 19.0. Software (v.19.0; SPSS, Chicago, IL, USA). Means were compared using the
3. Results
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Duncan Test for p<0.05 and with an analysis of variance (ANOVA).
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3.1. Effect of Si in the formation and remobilization of the apoplast Zn pool After two weeks of complete nutrition, Zn was removed from the nutrient solution (day 0). At that time, the Zn concentration at the washed roots (total Zn except apoplastic Zn) of plants from Zn 0 Si (0.0-0.0) reported intermediate values between the two Si treatments (Figure 1, A). A continuous addition of 0.5 mM of Si (Zn 0 Si (0.5-0.5)) produced the lowest accumulation in the washed roots and the Si 1.0 mM (Zn 0 Si (1.0-1.0)) the highest. A continuous decrease with time was observed for both Zn 0 Si (0.0-0.0) and Zn 0 Si (1.0-1.0) till the last day of measurements. However, after day 1, plants of Zn 0 Si (0.5-0.5) treatment highly increased the Zn content. At day 4 plants treated with 0.5 mM
ACCEPTED MANUSCRIPT Si had more than two-fold higher Zn content than the rest of the treatments, which presented nearly equal values. B)
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A)
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Figure 1: Effect of Si treatments in Zn content in (A) washed roots (µmol.root ) and (B) 1 apoplast (µmol.root- ) after Zn deficiency.
Soybean plants supplied with 0.5 mM Si presented the highest accumulation of
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apoplastic Zn (Figure 1, B) at day 0, in comparison with the two other treatments. Later on, it started to decrease and the last day of measurement (day 4) these plants are the ones with the lowest Zn content in the apoplast. Both Zn 0 Si (0.0-0.0) and Zn 0 Si (1.0-1.0) showed a continuous decrease in
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this parameter, however, it is not as pronounced as for Zn 0 Si (0.5-0.5).
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3.2. Long term Si effect on Zn deficient soybean plants Before Zn deficiency, no differences in SPAD values were obtained among treatments (data not shown). However, leaves and roots DW values (Figure 2) of Zn 10 Si (0.0) treatment were significantly lower in comparison with those of the two treatments with Si. Differences due to Si doses were only observed in stem DW, where higher values were obtained for 0.5 mM Si treated plants.
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Figure 2: Effect of Si treatments in leaves, stems and root dry weight (DW) (g) of soybean before the Zn deficiency period (M0). Different letters for the same vegetal organ denote significant differences according to Duncan test P<0.05.
The Zn content (Figure 3) in leaves and roots was extensively higher for plants treated with Si. By contrast, in stems, the Zn 10 Si (0.0) treatment led to a significant increase in this parameter. Within the two Si concentrations assayed, plants with Si 0.5 mM accumulated more Zn in stems and roots, but no
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significant differences were observed in leaves.
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Figure 3: Effect of Si treatments in Zn content (µmol.plant ) in leaves, stems and roots of soybean before the Zn deficiency period (M0). Different letters for the same vegetal organ denote significant differences according to Duncan test P<0.05.
ACCEPTED MANUSCRIPT As expected, Si concentration values at M0 sampling depended on the amount supplied of this element. Plants with an addition of 0.5 or 1.0 mM of Si had a higher amount of Si both in leaves and roots, whereas plants without Si resulted in the lowest values. The Si root content of plants grown under the Zn 10 Si (1.0) (10.9 µmol Si.plant-1) was more than twice as high as the one of Zn 10 Si
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(0.5) (4.8 µmol Si.plant-1), whereas Zn 10 Si (0.0) plants had only 0.10 µmol plant-1 of Si. For leaves, the observed values of Si content in µmol.plant-1 were: 186.7 for Zn 10 Si (1.0), 136.7 for Zn 10 Si (0.5), and 18.5 for Zn 10 Si (0.0).
Afterwards Zn deficiency, visual symptoms were not observed in soybean
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plants during the period of stress. On the contrary, plants of all treatments showed an increase in the SPAD values after two (∆SPAD: M1-M3) and three (∆SPAD: M1-M3) weeks of Zn removal (Table 2). The ones with a lower
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increase were the plants without both Zn and Si, while the treatments Zn 0 Si (1.0-1.0) and Zn 0 Si (1.0-0.0) submitted the higher values. Although the differences in the ameliorative effect of Si on SPAD data turned less pronounced between M1 and M3, the rise observed continued to be greater for both treatments with 1.0 mM Si.
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Table 2: Data of SPAD index increment at the second (SPAD at day 6 –SPAD at day 12; M1M2) and the third week (SPAD at day 6 –SPAD at day 21; M1-M3) after Zn deficiency at leaf level 6.
Treatments
∆SPAD index 2 week after Zn deficiency
3rd week after Zn deficiency
Zn 0 Si(0.0-0.0) Zn 10 Si(0.0-0.0) Zn 0 Si(0.5-0.5) Zn 0 Si(1.0-1.0) Zn 0 Si(0.5-0.0) Zn 0 Si(1.0-0.0)
2.49 4.05 5.16 6.19 2.72 5.62
7.14 7.77 7.35 7.46 7.01 8.14
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Similar values of DW (leaves, stems and roots), stems length and number of nodes were found at the three samplings for all the plants regardless the treatments (data not shown). The Zn content in leaves at M1, M2 and M3 (Table 3) was the lowest for the plants treated with Zn 0 Si (0.0-0.0) and the highest for Zn 10 Si (0.0-0.0). Intermediate values were obtained for the plants grown with Si. At the first and second samplings (M1 and M2), plants with an initial addition
ACCEPTED MANUSCRIPT of Si 0.5 mM showed significantly superior values of this parameter in leaves. Nevertheless, roots of plants with 1.0 mM of Si, added throughout the whole experiment or only at the beginning, stored more Zn (Table 3). A similar effect was observed for stems, although less pronounced. In the last sampling (M3), Si addition did not produce a major accumulation of Zn in roots, relative to
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plants of Zn 0 Si (0.0-0.0) treatment. In leaves and stems, at M3, the pattern followed was the same observed at M1 and M2, although the amount of Si added did not seem to influence the Zn accumulation. -1
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Table 3: Zinc content (µmol.plant ) at the three sampling times (M1, M2 and M3) after Zn removal from the NS. Values within a column followed by different letters differ significantly (P<0.05, Duncan test).
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Treatments
M1
M2
M3
0,44
d
0,68
c
0,84
Zn 10 Si(0.0-0.0)
1,29
ab
1,25
a
5,26
Zn 0 Si(0.5-0.5)
1,12
bc
0,92
b
1,35
Zn 0 Si(1.0-1.0)
1,05
c
1,05
b
1,19
Zn 0 Si(0.5-0.0)
1,36
a
1,36
a
1,07
Zn 0 Si(1.0-0.0)
1,03
c
0,96
b
1,21
M1
M2
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Root Zn (μmol.root )
M3
M1
M2
0,057
c
0,165
b
0,270
c
0,12
d
0,24
b
0,37
b
a
0,075
bc
0,052
c
0,414
a
0,65
a
0,22
bc
2,73
a
b
0,083
bc
0,129
b
0,358
ab
0,14
d
0,15
d
0,49
b
bc
0,096
ab
0,250
a
0,415
a
0,27
bc
0,37
a
0,38
b
c
0,075
bc
0,165
b
0,278
c
0,18
cd
0,16
cd
0,56
b
bc
0,118
a
0,176
b
0,334
bc
0,38
b
0,17
cd
0,43
b
Plants without a Si supply, either under Zn deficiency or not, accumulated greater Si in roots (Table 4). Moreover, the content in stems was nearly
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unappreciated and leaves content was lower in comparison with those of the plants supplied with Si. A continuous addition of 0.5 mM Si produced a greater storage of Si in leaves and a decrease in its deposition in roots and stems. The
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M3
d
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Zn 0 Si(0.0-0.0)
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Stems Zn (μmol.stems )
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Leaves Zn (μmol.leaves )
same pattern was detected in plants grown with 1.0 mM dose of Si, initially or continuously, but the effect was less relevant.
ACCEPTED MANUSCRIPT Table 4: Percentage of Si in roots and shoots in respect to the total Si content in soybean treated with different Si doses. The data are given for tree of the samplings (M1, M2 and M3).
Percentage of Si Leaves 80.29 89.09 96.94 94.11 96.14 95.28
Root 31.21 25.07 1.59 7.17 3.24 3.94
Leaves Root 68.79 21.31 74.93 7.94 97.29 1.43 91.32 3.30 96.76 10.12 95.69 5.97
M3 Stems 0.00 0.00 1.29 1.34 0.63 0.00
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Root 18.73 10.91 1.93 4.46 3.39 2.65
M2 Stems 0.00 0.00 1.12 1.51 0.00 0.38
Leaves 78.69 92.06 97.28 95.36 89.25 94.03
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Treatments Zn 0 Si(0.0-0.0) Zn 10 Si(0.0-0.0) Zn 0 Si(0.5-0.5) Zn 0 Si(1.0-1.0) Zn 0 Si(0.5-0.0) Zn 0 Si(1.0-0.0)
M1 Stems 0.98 0.00 1.13 1.43 0.47 2.07
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4. Disscusion
4.1. Effect of Si in the formation and remobilization of the apoplast Zn pool on Zn deficient soybean plants
There are no studies available that had measured apoplast Zn content to check the effect of Si in relation to Zn limited conditions. Nevertheless, there are experiments that had assayed the formation of Fe pools in roots in Fe deficient
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plants treated with Si (i.e. Pavlovic et al., 2013), which results are similar to the ones we have obtained.
We have reported a highest presence of Zn in the apoplast of Zn 0 Si (0.5-0.0) plants before Zn deficiency (Figure 1). After the Zn removal, these plants
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remobilized and used the Zn accumulated in roots. This is why, Zn in the apoplast started to diminish at the very next moment the stress commenced
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and at the same time, there was observed an increase of this micronutrient content in the rest of the root. We proposed that, with 0.5 mM of Si, a major Zn pool is formed in the free space of the roots during the no-stress period which is better remobilized when needed. A similar behavior was reported by Pavlovic et al. (2013) related to Fe with an addition of 1.5 mM Si. They observed that Si had a role in alleviating Fe deficiency symptoms such as chlorosis alongside an increase of the apoplastic Fe pool in the roots. This increase was afterwards followed by a rapid decrease once the Fe of the nutrient solution was eliminated and it was accompanied by a significantly higher Fe concentration in xylem sap.
ACCEPTED MANUSCRIPT Although Bityutskii et al. (2014) measured metal-mobilizing compounds, such as carboxylates and phenolics, likely candidates for xylem transport of Fe and which biosynthesis might involve Si (Pavlovic et al., 2013; Rellán-Álvarez et al., 2010); they concluded that Si clearly alleviates Fe deficiency by an enhancement of Fe distribution towards shoots due to an accumulation of Fe
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mobilizing compounds. Gonzalo et al. (2013) also suggested that there had been formed a pool of Fe-Si in the root apoplast which could be the reason for the delay of the chlorosis symptoms and the positive effects they have seen in their experiment with cucumber and soybean.
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The explanation proposed for the Si role is that, under Fe deprived conditions, Si may act by enhancing the reutilization of apoplastic Fe, thereby improving Fe nutrition in shoot (Bityutskii et al., 2014; Jin et al., 2007; Pavlovic et al., 2013).
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Under physiological conditions, the Fe becomes unavailable to the plant because of the formation of aggregates of colloidal particles containing Fe-O (Iler, 1979). If Si is present, aggregation is prevented by the formation of a silicic acid coating around the particles, producing a structure similar to that of the soil mineral ferrihydrite (Perry and Keeling-Tucker, 1998), which could be available for plants.
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Hernández-Apaolaza (2014) made a resemblance between the mechanism thought for the Si action in plants under Fe deficiency and the possible one for Zn deprived plants. The results reported here seemed to support this theory.
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This author suggests that root Zn deposits could be used under these conditions through the activation of a Zn-efficiency mechanism. And as for Fe, the Zn pools could be more mobile when Si is added, contributing to a better
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distribution and use of the available Zn (Hernández-Apaolaza, 2014). This will help the plant to ameliorate the symptoms of Zn deficiency. There are discrepancies between the amounts of Si that would be the most effective. In this work, plants with a supply of 1.0 mM Si showed a very different behavior to the one described above for 0.5 mM Si. The latter treatment seems to maintain the Zn stored in the apoplast (Figure 1), which impaired a strong fall of this nutrition content in the rest of the root compartments under Zn sufficient conditions, but when needed it seems to be remobilized to the rest of the plant. For Bityutskii et al. (2014) and Pavlovic et al. (2013), 1.5 mM was the concentration of Si chosen and proved positive under Fe deficiency, although
ACCEPTED MANUSCRIPT they only tested this one and the experimental growth conditions and plant species were different to the ones we have applied. The most effective Si dose for soybean (Gonzalo et al., 2013) was 0.5 mM, added only at the initial steps of growth. They considered that a higher Si dose would not be positive because it could cause an equilibrium displacement due
low formation rate of the Zn-Si pool.
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to the high amount of Si added that removed Zn from solution, concluding in a
The increase of the apoplastic Zn storage under Si supply and further
term experiment was developed.
4.2. Si effect on Zn no deficient soybean plants
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mobilization needed to be proved in a further experiment this is why; the long
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In this experiment, plants grown under complete nutrient solution with Zn and three different Si doses showed differences in DW (Figure 2). The Si addition increased significantly soybean leaves, stems and root DW for 0.5 and 1.0 mM Si supplies. A similar effect was obtained for rice (Ma and Yamaji, 2006; Mehrabanjoubani et al., 2015), cucumber (Adatia and Besford, 1986) and cotton (Li et al., 1989). The Zn-Si association in plants has been studied mainly under
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Zn toxicity conditions (Neuman and De Figueiredo, 2002; Neuman and zur Nieiden, 2001; Song et al., 2011). In those studies, Zn-Si precipitates in plants cell walls and vacuoles, compounds that might be later on degraded (Neumann
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and De Figueiredo, 2002).
In our experimental conditions, where Zn was available for the plant but not excessively, an extensively higher accumulation of Zn in the leaves of the plants
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under the treatments Zn 10 Si (0.5) and Zn 10 Si (1.0) was observed (Figure 3), suggesting a role of Si in Zn transportation from roots to shoots. Thus, in no stressed conditions, the Zn-Si compound seems to have a positive effect in Zn transport within the plant. Accordingly, Zn reaches leaves easily, whereas if Zn content in the nutrient solution is toxic, the plant might protect itself by synthesizing Zn-Si precipitates (Neumann and De Figueiredo, 2002). This theory might not be consistent with the higher amount of Zn stored in stems for no Si treated plants (Figure 3), in comparison with the ones of Zn 10 Si (0.5) and Zn 10 Si (1.0). Nevertheless, provided Si is present in the nutrient solution, soybean plants transport or accumulate the Zn into those tissues
ACCEPTED MANUSCRIPT where would be more useful: leaves and roots. It is well known the narrow relation between Zn and the photosynthesis and nutrient uptake processes, which take place mainly in leaves and roots, respectively (Tsonev et al., 2012): Zn participates actively in the process of water absorption and transport (Disante et al., 2010), in the fixation of carbon dioxide, in the stomata opening
integrates the structure of Rubisco (Alloway, 2004).
4.3. Si effect on Zn deficient soybean plants
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(Sharma et al., 1995), in proteolytic activities (Hänsch and Mendel, 2009); or it
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Despite other studies (Mehrabanjoubani et al., 2015); in the present experiment there were no statistical differences in soybean biometrical parameters (dry weight, number of nodes, plant length, etc) during the whole experiment
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regardless treatments with or without Si application. Moreover, the increments of the SPAD parameter within the weeks of Zn deficiency were higher when Si was added to the nutrient solution which showed that the Si supply affected chlorophyll production. Zinc is an indispensable component of many enzymes, and a structural stabilizer of protein and plant membranes (Aravind and Prassad, 2004). Likewise, it assures a properly development and formation of
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the chloroplast and it is necessary for the production of pigments such us the chlorophyll (Klung, 1999). This is why, in numerous experiments, it has been found a decrease in the chlorophyll content in leaves in different types of
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cultivars under Zn deficient conditions (Kösesakal and Ünal, 2009). However, the SPAD index is not always an indicative of the lack of this pigment, especially at the first stages of the stress (Ferreira et al., 2013). Even no
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changes by the addition of Si were reported by Bityutskii et al. (2014) in specific measures of chlorophyll concentration in cucumber Zn deprived plants. By contrast, in our study, a more pronounced increment in the SPAD values was recorded in some of the highest nodes (level 6) for plants with a Si supply (Table 2). The effect was more significant at the first stages of the experiment (until the day 12 after the Zn elimination of the nutrient solution). The discrepancies with and among other studies are suggested to be produced due to the likely antioxidant action of Si (Bityutskii et al., 2014) and its participation in the activation of enzymatic systems that avoid the chlorophyll degradation (Gottardi et al., 2012); fact produced thanks to the inner positive
ACCEPTED MANUSCRIPT benefits of Si that are no related to Zn nutrition. That will be consistent to the fact that the treatments that achieved a greater ∆SPAD index in our experiment were the ones with a higher amount of Si added: Zn 0 Si (0.5-0.5), Zn 0 Si (1.00.0) and Zn 0 Si (1.0-1.0), especially at the second week after Zn deficiency (Table 2).
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Silicon treatments were found to increase Zn content in leaves of plants with no Zn in the nutritional solution. The plants grown under an initial supply of 0.5 mM Si were the ones that reached the highest values, similar to plants without Zn deficiency. The effect lasted mainly for the first two weeks of deficiency (Table
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3). Mehrabanjoubani et al. (2015) observed that Si nutrition displayed a positive effect on vegetative growth in all organs of rice plants under Zn deficiency. They thought that these positive effects might be attributed to the greater plant Zn
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content. Meanwhile, Bityutskii et al. (2014) did not observe a Si alleviation of Zn deficiency symptoms in cucumber, but prevented necrotic spots may be due to the Si indirect effect on the antioxidant defense capacity. This suggestion is supported by some demonstrations of the Si defensive action in soybean plants (Gong et al., 2005; Liang et al., 2008) and the activation of the oxidative answer
(Marschner, 1995).
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under Zn deficiency by the generation of ROS and the decrease in SOD activity
Nevertheless, the initial accumulation of Zn at the root apoplast of plants treated with Si 0.5 mM and its rapid decreased when Zn deficiency were imposed
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(Figure 1) suggested that this pool may play an important role on Zn deficiency symptoms alleviation by Si addition. The root apoplast is of particular importance for nutrient acquisition, because the epidermal cell walls contain
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negatively charged sites (e.g. pectic polysaccharides abundant in carboxylic groups), which can serve as storage for most cationic nutrients (Sattelmacher, 2001). On the other hand, polysilicate can fix metal ions as chelate-like complexes (Iler, 1979), which may also occur in the plant cell wall during polymerization of orthosilicic acid. Pavlovic et al. (2013) and Bityutskii et al. (2014) concluded that the supply of Si led to significantly higher Fe accumulation in the apoplast of cucumber roots, which successfully ameliorated leaf symptoms of Fe deficiency. As Hernández-Apaolaza (2014) suggested, the Si and Zn accumulation detected in leaves alongside the root pools, prompt a
ACCEPTED MANUSCRIPT remobilization of this micronutrient, which could contribute to a more effective alleviation of Zn deficient symptoms. Besides, the data of the percentage of Si in root and shoots obtained in this experiment (Table 4) reinforces the conception exposed: plants with an addition of 0.5 mM Si accumulated more Si in leaves at M1, M2 and M3, which are the
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ones with a greater content of Zn in leaves (Table 3). They also generated a more abundant storage of Zn in the apoplast at the first days of Zn deficiency (Figure 1). Furthermore, for Zn 0 Si (0.5-0.5) plants, we can observe how the Zn pool is remobilized immediately only two days after de deficiency since the Zn
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content decrease in this root compartment. The interaction between Zn and Si uptake differs among literature. Bityutskii et al. (2014) shown that the total Si concentration in both roots and leaves was not affected by the Zn supply, while
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Mehrabanjoubani et al. (2015) reported that an increase of the Zn concentration added in the root medium led to increased Si concentration of root and shoot. Although, several studies have proved that both elements, Zn and Si presented a similar location in plants, which would favor a coordinate action (Gu et al.,
4.4. Conclusion
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2012; Hernández-Apaolaza, 2014; Neumann et al., 1997).
Due to the results acquired, we suggest a direct relation of Si with a major Zn accumulation in root apoplast and a more effective transport of Zn within
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soybean plants under Zn deficient conditions. Under a complete nutrition, an initial application of 0.5 mM Si produces a greater accumulation of Zn in root apoplast, which would be easily remove up to the leaves at the lack of Zn. This
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remobilization and distribution would prevent Zn deficiency symptoms. A continuously or a greater supply (1.0 mM) of Si would not be more beneficial.
Contributions
Acknowledgements Authors would like to thank H. Pérez Jordán and H. Hristov for their technical assistance and laboratory support. This work was supported by project AGL2013-44474-R from the Spanish Ministerio de Economía y Competitividad.
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ACCEPTED MANUSCRIPT Cakmak, I. 2000. Role of zinc in protecting plant cells from reactive oxygen species. New Phytol 146,185–205. Chen, C.C., Dixon, J.B., Turner, F.T. 1980. Iron coatings on rice roots: morphology and models of development. Soil Sci. Soc. Am. J. 44, 1113–1119.
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Gottardi, S., Iacuzzo, F., Tomasi, N., Cortella, G., Manzocco, L, Pinton, R., Römheld, V., Mimmo, T., Scanpicchio, M., Dalla Costa, L., Cesco, S. 2012. Beneficial effects of silicon on hydroponically grown corn salad (Valerianella locusta (L.) Laterr) plants. Plant Physiol. Biochem. 56, 14-23.
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Gu, H., Zhan, S.-S., Wang, S.-Z., Tang, Y.-T., Chanley, R-L., Fang, X.-H., Cai, X.-D., Qui, R.-L. 2012. Silicon-mediated amelioration of zinc toxicity in rice (Oryza sativa L.) seedlings. Plant Soil. 350, 193-204.
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Hänsch, R., Mendel, R.R. 2009. Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl), Curr. Opin. Plant Biol. 12, 259– 266. Hernández-Apaolaza, L. 2014. Can silicon partially alleviate micronutrient deficiency in plants? A Review. Planta 240, 447–458. Iler, R.K. 1979. The Chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry. Jonh Wiley & Sons. Wiley Interscience Publication. New York. Iwasaki, K., Maier, P., Fecht, M., Horst, W.J. 2002. Leaf apoplastic silicon enhances manganese tolerance of cowpea (Vigna unguiculata). J. Plant Physiol. 159, 167–173.
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Kösesakal, T., Ünal, M. 2009. Role of zinc deficiency in photosynthetic pigments and peroxidase activity of tomato seedlings. Journal of Biology 68, 113-120.
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Li, Y.C., Adva, A.K., Sumner, M.E. 1989. Response of cotton cultivars to aluminum in solutions with varying silicon concentrations. J. Plant Nutr. 12, 881–92.
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Liang, Y.C., Zhu, J., Li, Z.J., Chu, G.X., Ding, Y.F., Zhang, J., Sun, W.C. 2008 Role of silicon in enhancing resistance to freezing stress in two contrasting winter wheat cultivars. Environ Exp. Bot. 64, 286–294. Ma, J.F., Yamaji, N. 2006. Silicon uptake and accumulation in higher plants. Trends Plant Sci. 11, 392–397. Maksimovic, D., Mojovic, M., Maksimovic, V., Römheld, V., Nikolic, M. 2012. Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast. Journal of Exp. Bot. 63, 2411-2420.
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Marschner, H. 1995. Mineral Nutrition of Higher Plants. 2º ed. Academic Press. Mehrabanjoubani, P., Abdolzadeh, A., Sadeghipour, H.R., Aghdasi, M. 2015. Impacts of silicon nutrition on growth and nutrient status of rice plants grown under varying zinc regimes. Theoretical and Exp. Plant Phys. 27(1), 19-29.
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Neumann, D., Zur Nieden, U., Schwieger, W., Leopold, I., Lichtenberger, O., 1997. Heavy metal tolerance of Minuartia verna. J. Plant Physiol. 151, 101–108. Novozamsky I., van Eck R., Houba V.J.G. 1984. A rapid determination of silicon in plant material. Commun Soil Sci Plant Anal 15, 205–211. Pavlovic, J., Samardzic, J., Masimovic, V. Timotijevic, G., Stevic, N., Laursen, K.H., Hansen, T.h., Husted, S., Schjoerring, J.K., Liang, Y., Nikolic, M. 2013. Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast. New Phytol. 198, 1096-1107. Perry, C.C., Keeling-Tucker, T. 1998. Aspects of bioinorganic chemistry of silicon in conjunction with the biometals calcium, iron and aluminium. J. Inorg. Chem. 69, 181-191.
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Rellán-Álvarez, R., Giner-Martínez-Sierra, J., Orduna, J., Orera, I., RodríguezCastrillón, J.A., Carcía-Alonso, J.I., Abadía, J., Álvarez-Fernández, A., 2010. Identification of a tri-iron (III), tri-citrate complex in the xylem sap of irondeficient tomato resupplied with iron: new insights into plant iron long-distance transport. Plant Cell Physiol. 51, 91-102. Rengel, Z. 1999. Physiological reponses of wheat genotypes grown in chelatorbuffered nutrient solutions with increasing concentrations of excess HEDTA. Plant and Soil. 215, 193-202.
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Sattelmacher B. 2001. The apoplast and its significance for plant mineral nutrition. New Phytologist 149, 167–192.
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Sharma, P. N., Tripathi, T.T., Bisht, S.S. 1995. Zinc requirement for stomatal opening in cauliflower. Plant Physiol. 107, 751-756. Song, A.L., Li, P., Li, Z.J., Fan, F.L., Nikolic, M. 2011. The alliviaton of Zn toxicity by silicon is related to zinc transport and antioxidative reactions in rice. Plant Soil 344, 319-333. Tsonev, T., Cebola Lidon, F.J. 2012. Zinc in plants- An overview. Emir. J. Food Agric. 24 (4), 322-333.
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Van der Vorm, P.D.J. 1987. Dry ashing of plant material and dissolution of the ash in HF for the colorimetric determination of silicon. Commun Soil Sci. Plant Anal. 18, 1181–1189.
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Wu, J.-W., Shi, Y., Zhu, Y.-X., Wang, Y.-C., Gong, H.-J., 2013. Mechanisms of enhanced heavy metal tolerance in plants by silicon: a review. Pedosphere 23, 815–825.
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Zhu, Y., Gong, H., 2014. Beneficial effects of silicon on salt and drought tolerance inplants. Agron. Sustain. Dev. 34, 455–472. Zuluaga, J., Rodríguez, N., Rivas-Ramirez, I., de la Fuente, V., Rufo, L., Amils, R. 2011. An improved semiquantitative method for elemental analysis of plants using inductive coupled plasma mass spectrometry. Biol. Trace Elem. Res. 144, 1302-1317.
ACCEPTED MANUSCRIPT HIGHLIGHTS • Silicon effect in zinc deficiency symptoms alleviation and Zn distribution in soybean plants has been studied.
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• An initial and a continuous supply of silicon at the nutrient solution were tested.
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• An initial application of 0.5 mM of Si led to the highest root apoplast Zn accumulation and contributed to alleviate dry weight decrease due to the Zn deficiency and improve the Zn plant distribution.
ACCEPTED MANUSCRIPT CONTRIBUTIONS TO THE MANUSCRIPT:
Mª BLANCA PASCUAL: plant growth and analysis, manuscript writing
VIRGINIA ECHEVARRIA: technical support to plant growth and analysis,
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Mª JOSÉ GONZALO: data revision and manuscript supervision
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LOURDES HERNÁNDEZ-APAOLAZA: experimental design, data revision and manuscript correction and final elaboration
S0981-9428(16)30275-3
DOI:
10.1016/j.plaphy.2016.07.008
Reference:
PLAPHY 4616
To appear in:
Plant Physiology and Biochemistry
Received Date: 19 April 2016 Revised Date:
6 July 2016
Accepted Date: 9 July 2016
Please cite this article as: M.B. Pascual, V. Echevarria, M.J. Gonzalo, L. Hernández-Apaolaza, Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency, Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.07.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Silicon addition to soybean (Glycine max L.) plants alleviate zinc deficiency
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Mª BLANCA PASCUAL, VIRGINIA ECHEVARRIA, Mª JOSÉ GONZALO, LOURDES HERNÁNDEZ-APAOLAZA Agricultural Chemistry and Bromatology Department, Francisco Tomas y Valiente Street, n 7. Autonoma University 28049, Madrid, Spain [email protected] [email protected] [email protected]
Abstract
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Corresponding Author: Lourdes Hernández-Apaolaza [email protected] Phone: +34 914976859 Fax: +34 914973825
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[email protected]
It is well established the beneficial role of silicon (Si) in alleviating abiotic stress. However, it remains poorly understood the mechanisms of the Si-mediated
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protection against metal deficiency, especially the zinc (Zn) one. Recently, it has been proposed that Si may act by an interaction with this biometal in the root apoplast contributing to its movement through the plant, as in the case of Fe deficiency. In the present work, the effect of initial or continuous Si doses in
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soybean Zn deficient plants has been studied. For that purpose, plants grown in hydroponic culture were treated with different Si doses (0.0, 0.5 and 1.0 mM)
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under Zn limiting conditions. SPAD index in leaves, several growth parameters, mineral content in the whole plant and the formation of Zn pools in roots were determined. An initial addition of 0.5 mM of Si to the nutrient solution led to an enhancement of plants growth, Zn and Si content in leaves, and a higher storage of Zn in the root apoplast. The results suggest that this treatment enhanced Zn accumulation on roots and its movement to shoots when needed, mitigating Zn deficiency symptoms.
Key words: apoplast; silicon; soybean; zinc deficiency; Zn-Si interaction.
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1. Introduction Zinc (Zn) deficiency cause important disturbances in growth and development of in plants due to the large diversity of essential cellular functions and
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metabolic pathways directly influenced (Cakmak, 2000). Severe symptoms such as intervenial chlorosis in leaves, reddish-brown or bronze tints, epinasty, internode shortening, inward curling of leaf lamina and reductions in leaf size have been associated to Zn deficiency (Marschner, 1995).
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Although silicon (Si) is the second most abundant element in the earth’s crust, it is not still considered an essential element for higher plants. However, it has
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been largely known its beneficial effects alleviating various biotic (diseases, pests) and abiotic (salt and metal toxicity, high temperature, drought, freezing) stresses in many plant species (Epstein, 1999; Maksimovic et al., 2012; Romero et al., 2011; Song et al., 2011; Wu et al., 2013; Zhu and Gong, 2014). Moreover, in the past years there has been a rapid progress in the elucidation of how Si mediates under plant metal deficiency (Bityutskii et al. 2014; Gonzalo
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et al., 2013; Hernández-Apaolaza, 2014; Mehrabanjoubani et al., 2015; Pavlovic et al., 2013). However, despite the information available, the mechanisms of Si-mediated alleviation of nutrient deficiency in crops remain poorly understood.
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There are several evidences that Zn distribution in plant changed by the Si addition in hydroponic and soil experiments (Bityutskii et al., 2014; Gu et al.
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2012) and that both elements presented a similar location in plants (Gu et al., 2012). By using a fractionation technique, it has been shown that the cell wall bound fraction of Zn in roots, stems, sheaths and leaves of rice seedlings increased after Si addition (Gu et al., 2012). Moreover, in Fe plaque, Zn could be adsorbed (Chen et al. 1980) and remobilized when needed. Root Zn deposits could be used under Zn-deficient conditions through the activation of a Zn-deficiency mechanism (Hernández-Apaolaza, 2014). As for Fe (Pavlovic et al., 2013), the Zn apoplastic pools in the roots could be more mobile under deficiency conditions when Si was added, contributing to its better distribution inside plant (Hernández-Apaolaza, 2014). Recently, Bityutskii et al., (2014)
ACCEPTED MANUSCRIPT studied the effect of Si on Zn deficiency in acidic condition (nutrient solution pH 6.0) by growing cucumber seedlings without Zn and two Si doses (0 and 1.5 mM) in hydroponic culture. Even though no significant changes were observed in roots or leaves’ Zn and chlorophyll concentrations due to Si addition, Si partially diminished leaves necrotic spots. The reutilization of root apoplastic Fe
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via phenolics induced by Si nutrition did not appear under Zn deficiency and authors concluded that there were no evidences of Si alleviation of Zndeficiency symptoms, but as plants were grown without Zn in the nutrient solution, the possibility of Zn pools formation was suppressed, therefore their
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remobilization was not possible.
The aim of this work was to investigate the effect of Si addition on Zn deficient symptoms mitigation and the distribution of both elements, Zn and Si, in
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soybean seedlings under Zn limited conditions. In order to accomplish this objective different Si doses were applied to soybean plants grown in a nutrient solution with a deficient Zn content. The Zn deposits in apoplast were determined and its remobilization from root to shoot was also investigated.
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2. Materials and methods
2.1. Plant material and growing conditions Soybean seeds (Glycine max L. cv. Klaxon) were germinated on filter paper moistened with distilled water for one week in the dark at 28ºC. Thereafter,
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uniformly sized seeds were grown in aerated 1/5 strength nutrient solution for a week. Then, they were transferred to 2 L plastic buckets containing full-strength
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nutrient solution in which 0.2 g of solid CaCO3 were added per bucket. The composition of the nutrient solution was: macronutrients (mM) 1.0 Ca(NO3)2, 0.9 KNO3, 0.3 MgSO4, and 0.1 KH2PO4; micronutrient (µM) solution to avoid metal precipitations 35 NaCl, 10 H3BO3, 0.05 Na2MoO4, 50 Fe (III)-HBED, 10 ZnEDTA, 2.5 MnSO4, 1.0 CuSO4, 1.0 CoSO4, 1.0 NiCl2, and 115.5 EDTANa2 and 0.1 mM HEPES, 0.05 mM KOH. The solution was continuously aerated and renewed weekly. The pH was adjusted to 7.5 with NaOH 1.0 M or HCl 1.0 M. When necessary, the buckets were refilled with distilled water. Soybean plants were submitted to different Si concentrations in presence/absence of Zn depending on the experiments.
ACCEPTED MANUSCRIPT For the preparation of the Fe chelate solution, HBED (N,N'-bis(2-hydroxybencyl) ethilendiamine-N,N'diacetic acid, Strem Chemicals) was dissolved in sufficient NaOH (1/3 molar ratio) and then complexed with FeCl3·6H2O (Merck). After adjusting to 7.0 its pH, the solution was left to stand overnight to allow excess Fe to precipitate. Finally, the solution was filtered through 0.45 µm Millipore
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membrane and made up to volume. The Zn was supplied as Zn-EDTA, which was prepared by mixing at 1/1 molar ratio Zn(NO3)2·6H2O (98%. Sigma-Aldrich) and ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich).
Plants were grown under controlled conditions in a Dycometal-type CCK growth chamber provided with fluorescent and sodium vapor lamps with a 350 µE m−2
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s−1 light intensity, photoperiod 16/8 h, temperature (day/night) 30/20°C and
2.2. Experimental design.
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50/70% relative humidity.
2.2.1. Effect of Si in the formation and remobilization of the apoplast Zn pool Soybean plants, grown as mentioned above, were cultivated with a full strength nutrient solution for two weeks. Three different Si doses were added: 0.0, 0.5 and 1.0 mM as Na2SiO3·5H2O (Suprapur, Merk). Then, Zn supply was
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eliminated from all the nutrient solutions except for the plants of the control treatment and the Si was maintained in half of the plants (continuous silicon supply) for four more days. Table 1 gathers the six different treatments that
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were carried out.
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Table 1: Silicon and Zinc treatments (mM and µM, respectively) regarding to the days after treatments application (DAT).
Si (mM)
Zn (µM)
0-14 DAT
14-18 DAT
0-14 DAT
14-18 DAT
Abbreviation
0.0
0.0
0
0
Zn 0 Si (0.0-0.0)
0.0
0.0
10
10
Zn10 Si(0.0-0.0)
0.5
0.0
10
0
Zn 0 Si(0.5-0.0)
0.5
0.5
10
0
Zn 0 Si(0.5-0.5)
1.0
0.0
10
0
Zn 0 Si(1.0-0.0)
1.0
1.0
10
0
Zn 0 Si(1.0-1.0)
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hydroponic experiment lasted a total of five weeks.
2.3. Measurements.
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weeks after the Zn removal from the nutrient solution. Therefore, this
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In the first experiment described, the root apoplastic accumulation of Zn was tested after Zn removal from the nutrient solution. The method used was the one proposed by Rengel (1999) in which intact roots were washed twice in a
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mixture of distilled water and ice for 5 minutes each, and then with a solution containing 2 mM CaCl2 and 1 mM LaCl3 for 10 minutes in continue stirring. This last washed solution was filtrated, made up to 50 ml and Zn concentration was assessed by atomic absorption spectrophotometer (AAs, Perkin-Elmer Analyst 800). In addition, the washed roots were oven dried, and Zn content was measured after a microwave (CEM Corporation MARS 240/50; Matthews, NC,
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USA) digestion following the method described by Zuluaga et al. (2011) to determine the total amount of Zn in roots and its distribution. For that, 500 mg of dry material were loaded into Teflon vessels with 8 mL HNO3 at 65%, 2 mL
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H2O2 at 30% and 1 mL HF at 40% (all of them Suprapure reagent grade, Merck Millipore). After the digestion the samples were made up to 50 mL volume with distilled water and Zn concentration was determined by AAs.
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During the whole long term experiment, the SPAD index was monitored every three days using a digital chlorophyll meter SPAD (Soil and Plant Analyzer Development) model 502 (Minolta Co., Osaka, Japan). Samples of leaves, stems and roots of three plants per treatment were taken at 0(M0), 7 (M1), 14(M2) and 21 (M3) days before the Zn removal of the nutrition solution and fresh weight (FW) was determined. Immediately after the sampling, plants were washed with four different solutions: Tween 80 at 0.1%, 0.1 M of HCl (Suprapure, Merck Millipore), water and distilled water. Then, the biometric parameters dry weight (DW), stems length (SL), roots length (RL), and node
ACCEPTED MANUSCRIPT number (NN) were measured. In addition, a sample of all the nutrient solutions was taken before every change for their pH measurement and nutrient analysis. Fe, Cu, Mn, and Zn concentrations were determined in leaves, stems and roots following the previously described microwave method. Silicon was determined by digesting 20 mg of ground plant materials in 1.0 ml of
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the mixture of 1 M HCl and 2.3 M HF (v/v = 1/2) and shaken overnight. The samples were centrifuged at 10 g for 10 minutes, and 0.5 ml of the supernatant was added to plastic tubes. Afterwards, 3.0 ml of 20% glacial acetic acid and 1.0 ml of 54 g.L-1 (NH4)6Mo7O24·4H2O were added and incubated for 5 minutes,
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followed by addition of 0.5 ml of 20 g.L-1 tartaric acid and 2.0 ml reductant. Thirty minutes after the reductant addition, the sample absorbance was measured at 650 nm with a spectrophotometer (Jasco V-650). The reductant
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was a mixture of solution A: 80 g.L-1 Na2SO3 + 16 g.L-1 1-amino-2-naphthol-4sulfoacid and solution B: 12.5 g.L-1 NaHSO3. Then, the solution A and B were mixed together and made up to 250 ml (Iwasaki et al., 2002; Novozamsky et al., 1984, Van der Vorm, 1987)
2.4. Statistical analysis
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Obtained data were analyzed by using the IBM SPSS Statistics Version 19.0. Software (v.19.0; SPSS, Chicago, IL, USA). Means were compared using the
3. Results
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Duncan Test for p<0.05 and with an analysis of variance (ANOVA).
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3.1. Effect of Si in the formation and remobilization of the apoplast Zn pool After two weeks of complete nutrition, Zn was removed from the nutrient solution (day 0). At that time, the Zn concentration at the washed roots (total Zn except apoplastic Zn) of plants from Zn 0 Si (0.0-0.0) reported intermediate values between the two Si treatments (Figure 1, A). A continuous addition of 0.5 mM of Si (Zn 0 Si (0.5-0.5)) produced the lowest accumulation in the washed roots and the Si 1.0 mM (Zn 0 Si (1.0-1.0)) the highest. A continuous decrease with time was observed for both Zn 0 Si (0.0-0.0) and Zn 0 Si (1.0-1.0) till the last day of measurements. However, after day 1, plants of Zn 0 Si (0.5-0.5) treatment highly increased the Zn content. At day 4 plants treated with 0.5 mM
ACCEPTED MANUSCRIPT Si had more than two-fold higher Zn content than the rest of the treatments, which presented nearly equal values. B)
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A)
-1
Figure 1: Effect of Si treatments in Zn content in (A) washed roots (µmol.root ) and (B) 1 apoplast (µmol.root- ) after Zn deficiency.
Soybean plants supplied with 0.5 mM Si presented the highest accumulation of
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apoplastic Zn (Figure 1, B) at day 0, in comparison with the two other treatments. Later on, it started to decrease and the last day of measurement (day 4) these plants are the ones with the lowest Zn content in the apoplast. Both Zn 0 Si (0.0-0.0) and Zn 0 Si (1.0-1.0) showed a continuous decrease in
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this parameter, however, it is not as pronounced as for Zn 0 Si (0.5-0.5).
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3.2. Long term Si effect on Zn deficient soybean plants Before Zn deficiency, no differences in SPAD values were obtained among treatments (data not shown). However, leaves and roots DW values (Figure 2) of Zn 10 Si (0.0) treatment were significantly lower in comparison with those of the two treatments with Si. Differences due to Si doses were only observed in stem DW, where higher values were obtained for 0.5 mM Si treated plants.
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Figure 2: Effect of Si treatments in leaves, stems and root dry weight (DW) (g) of soybean before the Zn deficiency period (M0). Different letters for the same vegetal organ denote significant differences according to Duncan test P<0.05.
The Zn content (Figure 3) in leaves and roots was extensively higher for plants treated with Si. By contrast, in stems, the Zn 10 Si (0.0) treatment led to a significant increase in this parameter. Within the two Si concentrations assayed, plants with Si 0.5 mM accumulated more Zn in stems and roots, but no
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significant differences were observed in leaves.
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Figure 3: Effect of Si treatments in Zn content (µmol.plant ) in leaves, stems and roots of soybean before the Zn deficiency period (M0). Different letters for the same vegetal organ denote significant differences according to Duncan test P<0.05.
ACCEPTED MANUSCRIPT As expected, Si concentration values at M0 sampling depended on the amount supplied of this element. Plants with an addition of 0.5 or 1.0 mM of Si had a higher amount of Si both in leaves and roots, whereas plants without Si resulted in the lowest values. The Si root content of plants grown under the Zn 10 Si (1.0) (10.9 µmol Si.plant-1) was more than twice as high as the one of Zn 10 Si
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(0.5) (4.8 µmol Si.plant-1), whereas Zn 10 Si (0.0) plants had only 0.10 µmol plant-1 of Si. For leaves, the observed values of Si content in µmol.plant-1 were: 186.7 for Zn 10 Si (1.0), 136.7 for Zn 10 Si (0.5), and 18.5 for Zn 10 Si (0.0).
Afterwards Zn deficiency, visual symptoms were not observed in soybean
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plants during the period of stress. On the contrary, plants of all treatments showed an increase in the SPAD values after two (∆SPAD: M1-M3) and three (∆SPAD: M1-M3) weeks of Zn removal (Table 2). The ones with a lower
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increase were the plants without both Zn and Si, while the treatments Zn 0 Si (1.0-1.0) and Zn 0 Si (1.0-0.0) submitted the higher values. Although the differences in the ameliorative effect of Si on SPAD data turned less pronounced between M1 and M3, the rise observed continued to be greater for both treatments with 1.0 mM Si.
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Table 2: Data of SPAD index increment at the second (SPAD at day 6 –SPAD at day 12; M1M2) and the third week (SPAD at day 6 –SPAD at day 21; M1-M3) after Zn deficiency at leaf level 6.
Treatments
∆SPAD index 2 week after Zn deficiency
3rd week after Zn deficiency
Zn 0 Si(0.0-0.0) Zn 10 Si(0.0-0.0) Zn 0 Si(0.5-0.5) Zn 0 Si(1.0-1.0) Zn 0 Si(0.5-0.0) Zn 0 Si(1.0-0.0)
2.49 4.05 5.16 6.19 2.72 5.62
7.14 7.77 7.35 7.46 7.01 8.14
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nd
Similar values of DW (leaves, stems and roots), stems length and number of nodes were found at the three samplings for all the plants regardless the treatments (data not shown). The Zn content in leaves at M1, M2 and M3 (Table 3) was the lowest for the plants treated with Zn 0 Si (0.0-0.0) and the highest for Zn 10 Si (0.0-0.0). Intermediate values were obtained for the plants grown with Si. At the first and second samplings (M1 and M2), plants with an initial addition
ACCEPTED MANUSCRIPT of Si 0.5 mM showed significantly superior values of this parameter in leaves. Nevertheless, roots of plants with 1.0 mM of Si, added throughout the whole experiment or only at the beginning, stored more Zn (Table 3). A similar effect was observed for stems, although less pronounced. In the last sampling (M3), Si addition did not produce a major accumulation of Zn in roots, relative to
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plants of Zn 0 Si (0.0-0.0) treatment. In leaves and stems, at M3, the pattern followed was the same observed at M1 and M2, although the amount of Si added did not seem to influence the Zn accumulation. -1
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Table 3: Zinc content (µmol.plant ) at the three sampling times (M1, M2 and M3) after Zn removal from the NS. Values within a column followed by different letters differ significantly (P<0.05, Duncan test).
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Treatments
M1
M2
M3
0,44
d
0,68
c
0,84
Zn 10 Si(0.0-0.0)
1,29
ab
1,25
a
5,26
Zn 0 Si(0.5-0.5)
1,12
bc
0,92
b
1,35
Zn 0 Si(1.0-1.0)
1,05
c
1,05
b
1,19
Zn 0 Si(0.5-0.0)
1,36
a
1,36
a
1,07
Zn 0 Si(1.0-0.0)
1,03
c
0,96
b
1,21
M1
M2
-1
Root Zn (μmol.root )
M3
M1
M2
0,057
c
0,165
b
0,270
c
0,12
d
0,24
b
0,37
b
a
0,075
bc
0,052
c
0,414
a
0,65
a
0,22
bc
2,73
a
b
0,083
bc
0,129
b
0,358
ab
0,14
d
0,15
d
0,49
b
bc
0,096
ab
0,250
a
0,415
a
0,27
bc
0,37
a
0,38
b
c
0,075
bc
0,165
b
0,278
c
0,18
cd
0,16
cd
0,56
b
bc
0,118
a
0,176
b
0,334
bc
0,38
b
0,17
cd
0,43
b
Plants without a Si supply, either under Zn deficiency or not, accumulated greater Si in roots (Table 4). Moreover, the content in stems was nearly
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unappreciated and leaves content was lower in comparison with those of the plants supplied with Si. A continuous addition of 0.5 mM Si produced a greater storage of Si in leaves and a decrease in its deposition in roots and stems. The
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M3
d
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Zn 0 Si(0.0-0.0)
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Stems Zn (μmol.stems )
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Leaves Zn (μmol.leaves )
same pattern was detected in plants grown with 1.0 mM dose of Si, initially or continuously, but the effect was less relevant.
ACCEPTED MANUSCRIPT Table 4: Percentage of Si in roots and shoots in respect to the total Si content in soybean treated with different Si doses. The data are given for tree of the samplings (M1, M2 and M3).
Percentage of Si Leaves 80.29 89.09 96.94 94.11 96.14 95.28
Root 31.21 25.07 1.59 7.17 3.24 3.94
Leaves Root 68.79 21.31 74.93 7.94 97.29 1.43 91.32 3.30 96.76 10.12 95.69 5.97
M3 Stems 0.00 0.00 1.29 1.34 0.63 0.00
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Root 18.73 10.91 1.93 4.46 3.39 2.65
M2 Stems 0.00 0.00 1.12 1.51 0.00 0.38
Leaves 78.69 92.06 97.28 95.36 89.25 94.03
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Treatments Zn 0 Si(0.0-0.0) Zn 10 Si(0.0-0.0) Zn 0 Si(0.5-0.5) Zn 0 Si(1.0-1.0) Zn 0 Si(0.5-0.0) Zn 0 Si(1.0-0.0)
M1 Stems 0.98 0.00 1.13 1.43 0.47 2.07
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4. Disscusion
4.1. Effect of Si in the formation and remobilization of the apoplast Zn pool on Zn deficient soybean plants
There are no studies available that had measured apoplast Zn content to check the effect of Si in relation to Zn limited conditions. Nevertheless, there are experiments that had assayed the formation of Fe pools in roots in Fe deficient
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plants treated with Si (i.e. Pavlovic et al., 2013), which results are similar to the ones we have obtained.
We have reported a highest presence of Zn in the apoplast of Zn 0 Si (0.5-0.0) plants before Zn deficiency (Figure 1). After the Zn removal, these plants
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remobilized and used the Zn accumulated in roots. This is why, Zn in the apoplast started to diminish at the very next moment the stress commenced
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and at the same time, there was observed an increase of this micronutrient content in the rest of the root. We proposed that, with 0.5 mM of Si, a major Zn pool is formed in the free space of the roots during the no-stress period which is better remobilized when needed. A similar behavior was reported by Pavlovic et al. (2013) related to Fe with an addition of 1.5 mM Si. They observed that Si had a role in alleviating Fe deficiency symptoms such as chlorosis alongside an increase of the apoplastic Fe pool in the roots. This increase was afterwards followed by a rapid decrease once the Fe of the nutrient solution was eliminated and it was accompanied by a significantly higher Fe concentration in xylem sap.
ACCEPTED MANUSCRIPT Although Bityutskii et al. (2014) measured metal-mobilizing compounds, such as carboxylates and phenolics, likely candidates for xylem transport of Fe and which biosynthesis might involve Si (Pavlovic et al., 2013; Rellán-Álvarez et al., 2010); they concluded that Si clearly alleviates Fe deficiency by an enhancement of Fe distribution towards shoots due to an accumulation of Fe
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mobilizing compounds. Gonzalo et al. (2013) also suggested that there had been formed a pool of Fe-Si in the root apoplast which could be the reason for the delay of the chlorosis symptoms and the positive effects they have seen in their experiment with cucumber and soybean.
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The explanation proposed for the Si role is that, under Fe deprived conditions, Si may act by enhancing the reutilization of apoplastic Fe, thereby improving Fe nutrition in shoot (Bityutskii et al., 2014; Jin et al., 2007; Pavlovic et al., 2013).
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Under physiological conditions, the Fe becomes unavailable to the plant because of the formation of aggregates of colloidal particles containing Fe-O (Iler, 1979). If Si is present, aggregation is prevented by the formation of a silicic acid coating around the particles, producing a structure similar to that of the soil mineral ferrihydrite (Perry and Keeling-Tucker, 1998), which could be available for plants.
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Hernández-Apaolaza (2014) made a resemblance between the mechanism thought for the Si action in plants under Fe deficiency and the possible one for Zn deprived plants. The results reported here seemed to support this theory.
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This author suggests that root Zn deposits could be used under these conditions through the activation of a Zn-efficiency mechanism. And as for Fe, the Zn pools could be more mobile when Si is added, contributing to a better
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distribution and use of the available Zn (Hernández-Apaolaza, 2014). This will help the plant to ameliorate the symptoms of Zn deficiency. There are discrepancies between the amounts of Si that would be the most effective. In this work, plants with a supply of 1.0 mM Si showed a very different behavior to the one described above for 0.5 mM Si. The latter treatment seems to maintain the Zn stored in the apoplast (Figure 1), which impaired a strong fall of this nutrition content in the rest of the root compartments under Zn sufficient conditions, but when needed it seems to be remobilized to the rest of the plant. For Bityutskii et al. (2014) and Pavlovic et al. (2013), 1.5 mM was the concentration of Si chosen and proved positive under Fe deficiency, although
ACCEPTED MANUSCRIPT they only tested this one and the experimental growth conditions and plant species were different to the ones we have applied. The most effective Si dose for soybean (Gonzalo et al., 2013) was 0.5 mM, added only at the initial steps of growth. They considered that a higher Si dose would not be positive because it could cause an equilibrium displacement due
low formation rate of the Zn-Si pool.
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to the high amount of Si added that removed Zn from solution, concluding in a
The increase of the apoplastic Zn storage under Si supply and further
term experiment was developed.
4.2. Si effect on Zn no deficient soybean plants
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mobilization needed to be proved in a further experiment this is why; the long
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In this experiment, plants grown under complete nutrient solution with Zn and three different Si doses showed differences in DW (Figure 2). The Si addition increased significantly soybean leaves, stems and root DW for 0.5 and 1.0 mM Si supplies. A similar effect was obtained for rice (Ma and Yamaji, 2006; Mehrabanjoubani et al., 2015), cucumber (Adatia and Besford, 1986) and cotton (Li et al., 1989). The Zn-Si association in plants has been studied mainly under
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Zn toxicity conditions (Neuman and De Figueiredo, 2002; Neuman and zur Nieiden, 2001; Song et al., 2011). In those studies, Zn-Si precipitates in plants cell walls and vacuoles, compounds that might be later on degraded (Neumann
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and De Figueiredo, 2002).
In our experimental conditions, where Zn was available for the plant but not excessively, an extensively higher accumulation of Zn in the leaves of the plants
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under the treatments Zn 10 Si (0.5) and Zn 10 Si (1.0) was observed (Figure 3), suggesting a role of Si in Zn transportation from roots to shoots. Thus, in no stressed conditions, the Zn-Si compound seems to have a positive effect in Zn transport within the plant. Accordingly, Zn reaches leaves easily, whereas if Zn content in the nutrient solution is toxic, the plant might protect itself by synthesizing Zn-Si precipitates (Neumann and De Figueiredo, 2002). This theory might not be consistent with the higher amount of Zn stored in stems for no Si treated plants (Figure 3), in comparison with the ones of Zn 10 Si (0.5) and Zn 10 Si (1.0). Nevertheless, provided Si is present in the nutrient solution, soybean plants transport or accumulate the Zn into those tissues
ACCEPTED MANUSCRIPT where would be more useful: leaves and roots. It is well known the narrow relation between Zn and the photosynthesis and nutrient uptake processes, which take place mainly in leaves and roots, respectively (Tsonev et al., 2012): Zn participates actively in the process of water absorption and transport (Disante et al., 2010), in the fixation of carbon dioxide, in the stomata opening
integrates the structure of Rubisco (Alloway, 2004).
4.3. Si effect on Zn deficient soybean plants
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(Sharma et al., 1995), in proteolytic activities (Hänsch and Mendel, 2009); or it
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Despite other studies (Mehrabanjoubani et al., 2015); in the present experiment there were no statistical differences in soybean biometrical parameters (dry weight, number of nodes, plant length, etc) during the whole experiment
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regardless treatments with or without Si application. Moreover, the increments of the SPAD parameter within the weeks of Zn deficiency were higher when Si was added to the nutrient solution which showed that the Si supply affected chlorophyll production. Zinc is an indispensable component of many enzymes, and a structural stabilizer of protein and plant membranes (Aravind and Prassad, 2004). Likewise, it assures a properly development and formation of
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the chloroplast and it is necessary for the production of pigments such us the chlorophyll (Klung, 1999). This is why, in numerous experiments, it has been found a decrease in the chlorophyll content in leaves in different types of
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cultivars under Zn deficient conditions (Kösesakal and Ünal, 2009). However, the SPAD index is not always an indicative of the lack of this pigment, especially at the first stages of the stress (Ferreira et al., 2013). Even no
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changes by the addition of Si were reported by Bityutskii et al. (2014) in specific measures of chlorophyll concentration in cucumber Zn deprived plants. By contrast, in our study, a more pronounced increment in the SPAD values was recorded in some of the highest nodes (level 6) for plants with a Si supply (Table 2). The effect was more significant at the first stages of the experiment (until the day 12 after the Zn elimination of the nutrient solution). The discrepancies with and among other studies are suggested to be produced due to the likely antioxidant action of Si (Bityutskii et al., 2014) and its participation in the activation of enzymatic systems that avoid the chlorophyll degradation (Gottardi et al., 2012); fact produced thanks to the inner positive
ACCEPTED MANUSCRIPT benefits of Si that are no related to Zn nutrition. That will be consistent to the fact that the treatments that achieved a greater ∆SPAD index in our experiment were the ones with a higher amount of Si added: Zn 0 Si (0.5-0.5), Zn 0 Si (1.00.0) and Zn 0 Si (1.0-1.0), especially at the second week after Zn deficiency (Table 2).
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Silicon treatments were found to increase Zn content in leaves of plants with no Zn in the nutritional solution. The plants grown under an initial supply of 0.5 mM Si were the ones that reached the highest values, similar to plants without Zn deficiency. The effect lasted mainly for the first two weeks of deficiency (Table
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3). Mehrabanjoubani et al. (2015) observed that Si nutrition displayed a positive effect on vegetative growth in all organs of rice plants under Zn deficiency. They thought that these positive effects might be attributed to the greater plant Zn
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content. Meanwhile, Bityutskii et al. (2014) did not observe a Si alleviation of Zn deficiency symptoms in cucumber, but prevented necrotic spots may be due to the Si indirect effect on the antioxidant defense capacity. This suggestion is supported by some demonstrations of the Si defensive action in soybean plants (Gong et al., 2005; Liang et al., 2008) and the activation of the oxidative answer
(Marschner, 1995).
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under Zn deficiency by the generation of ROS and the decrease in SOD activity
Nevertheless, the initial accumulation of Zn at the root apoplast of plants treated with Si 0.5 mM and its rapid decreased when Zn deficiency were imposed
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(Figure 1) suggested that this pool may play an important role on Zn deficiency symptoms alleviation by Si addition. The root apoplast is of particular importance for nutrient acquisition, because the epidermal cell walls contain
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negatively charged sites (e.g. pectic polysaccharides abundant in carboxylic groups), which can serve as storage for most cationic nutrients (Sattelmacher, 2001). On the other hand, polysilicate can fix metal ions as chelate-like complexes (Iler, 1979), which may also occur in the plant cell wall during polymerization of orthosilicic acid. Pavlovic et al. (2013) and Bityutskii et al. (2014) concluded that the supply of Si led to significantly higher Fe accumulation in the apoplast of cucumber roots, which successfully ameliorated leaf symptoms of Fe deficiency. As Hernández-Apaolaza (2014) suggested, the Si and Zn accumulation detected in leaves alongside the root pools, prompt a
ACCEPTED MANUSCRIPT remobilization of this micronutrient, which could contribute to a more effective alleviation of Zn deficient symptoms. Besides, the data of the percentage of Si in root and shoots obtained in this experiment (Table 4) reinforces the conception exposed: plants with an addition of 0.5 mM Si accumulated more Si in leaves at M1, M2 and M3, which are the
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ones with a greater content of Zn in leaves (Table 3). They also generated a more abundant storage of Zn in the apoplast at the first days of Zn deficiency (Figure 1). Furthermore, for Zn 0 Si (0.5-0.5) plants, we can observe how the Zn pool is remobilized immediately only two days after de deficiency since the Zn
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content decrease in this root compartment. The interaction between Zn and Si uptake differs among literature. Bityutskii et al. (2014) shown that the total Si concentration in both roots and leaves was not affected by the Zn supply, while
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Mehrabanjoubani et al. (2015) reported that an increase of the Zn concentration added in the root medium led to increased Si concentration of root and shoot. Although, several studies have proved that both elements, Zn and Si presented a similar location in plants, which would favor a coordinate action (Gu et al.,
4.4. Conclusion
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2012; Hernández-Apaolaza, 2014; Neumann et al., 1997).
Due to the results acquired, we suggest a direct relation of Si with a major Zn accumulation in root apoplast and a more effective transport of Zn within
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soybean plants under Zn deficient conditions. Under a complete nutrition, an initial application of 0.5 mM Si produces a greater accumulation of Zn in root apoplast, which would be easily remove up to the leaves at the lack of Zn. This
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remobilization and distribution would prevent Zn deficiency symptoms. A continuously or a greater supply (1.0 mM) of Si would not be more beneficial.
Contributions
Acknowledgements Authors would like to thank H. Pérez Jordán and H. Hristov for their technical assistance and laboratory support. This work was supported by project AGL2013-44474-R from the Spanish Ministerio de Economía y Competitividad.
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ACCEPTED MANUSCRIPT HIGHLIGHTS • Silicon effect in zinc deficiency symptoms alleviation and Zn distribution in soybean plants has been studied.
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• An initial and a continuous supply of silicon at the nutrient solution were tested.
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• An initial application of 0.5 mM of Si led to the highest root apoplast Zn accumulation and contributed to alleviate dry weight decrease due to the Zn deficiency and improve the Zn plant distribution.
ACCEPTED MANUSCRIPT CONTRIBUTIONS TO THE MANUSCRIPT:
Mª BLANCA PASCUAL: plant growth and analysis, manuscript writing
VIRGINIA ECHEVARRIA: technical support to plant growth and analysis,
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Mª JOSÉ GONZALO: data revision and manuscript supervision
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LOURDES HERNÁNDEZ-APAOLAZA: experimental design, data revision and manuscript correction and final elaboration