Dynamic of monosilicic and polysilicic acids in plant tissues under salt stress conditions

Matichenkov V.V.*, Biel K.Y. **, Bocharnikova E.A. ***

* Institute Basic Biological Problems Russian Academy of Sciences, Pushchino, Russia.

E-mail: vvmatichenkov@rambler.ru
** Biosphere Systems International, Tucson, Arizona 85755,USA
*** Institute Physical-Chemical and Biological Problems in Soil Science Russian
Academy of Sciences, Russia. E-mail: mswk@rambler.ru

The problem of salt toxicity is one of the most pressing problems in modern agriculture,
and every year we see this condition getting worse, especially with increased global
warming. As it is well known from scientific literature, plants can employ silicon as
protective agent against stresses. However, the absence of methods for the determination
of the presence of soluble forms of Si in plant’s tissue hinders the progress in
understanding the mechanisms of this process. We suggest two methods for quantifying
monosilicic and polysilicic acids in plant sap, in apoplast and symplast of plant tissues.
The realization of these methods was conducted in vegetative experiments with barley as
test plant.
Plants grew in media low in plant-available Si. Plants were cut near the root level and put
into containers with water having 550 mM NaCl solutions or kept without water. Samples
of leaf blades and internodes of stems were collected at the following intervals: just after
cutting, at 24 hours and at 48 hours after cutting. The contents of monosilicic acid and
polysilicic acid were determined in fresh plant tissues and separately in apoplast and
symplast of plant tissues. Total plant Si was analyzed as well. The content of monosilicic
acid in the sap of barley plants ranged from 195.2 to 368.2 Si mg/kg of fresh weight of
the plant. The maximum concentrations were found in the roots and the minimum
concentrations in the internodes of stems. The content of polysilicic acid varied from 363
to 682 mg/kg of fresh weight of the plant. The maximum concentrations of polysilicic
acid were determined in the roots and the minimum concentrations in the noodles of
stems. Under salt stress conditions, the soluble form of Si in plant tissues increased and
this can be explained by surprising Si positioning within the plant. Our elaborated
methods make it possible to obtain new information about soluble silicon compounds in
the sap of plants. The separation of mono- and polysilicic acids in the sap and in apoplast
and symplast provides the understanding of this biochemical process, controlled by active
Si compounds.

Introduction
The total area of salt-affected land in the world is estimated to be approximately 397
million ha, of which 45 million ha are irrigated lands (FAO, 2008). Using of salt-bearing
water for irrigation in regions with deficiency of fresh water, results in increased
salinization of crop lands.

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 Cultivation in salt affected soils is only possible if salt is removed or by increasing
plant salt resistance. Besides gene-modification of plants to increase plant salt resistance,
it is also possible to achieve this goal by improving plant nutrition (Alsaadawi & Dahash,
2000; Maas, 1985). This is clearly shown in scientific studies in which plant resistance to
salt toxicity is enhanced when silicon (Si) fertilization is applied (Liang, 1999;
Kosobrukhov & Matichenkov, 2004).
Most virgin soils, except volcanic, alluvial and rich Mollisols, have deficiency of
plant-available Si forms (Matichenkov et al., 2001 a). Plants growing on sandy, degraded,
or over-used soils suffer from critical deficiency of this element and lack of Si plant
nutrition, resulting in decreased plant resistance to biotic and abiotic stresses
(Matichenkov et al., 2001b). The application of Si fertilizers or Si soil amendments
increases plant resistance to high salt content in soil (Liang, 1999, Kosobrukhov &.,
Matichenkov, 2004). There are several hypotheses explaining this effect, they are: (i)
improving photosynthetic activity, (ii) enhancing K:Na selectivity ratio, (iii) increasing
enzymatic activity, and (iv) increasing concentration of soluble substances in the xylem,
which results in reduced sodium adsorption by plants (Ahmad et al., 1992; Bradbury &
Ahmad , 1990; Iler, 1979). Increased monosilicic acid concentration in soil leads to
decreased plant-Na adsorption from the soil (Liang, 1999; Kosobrukhov & Matichenkov,
2004; Matichenkov et al., 2001a). Probably, the beneficial Si effect on plant’s salt
resistance is caused by the simultaneous action of several mechanisms. To elucidate and
understand these mechanisms is important for the elaboration of the technology that
increases plant resistance to salt toxicity.
Germaine was used in many of the experiments related with this investigation on salt
toxicity and Si effects on plants (Matichenkov & Kosobryukhov, 2003; Snyder et al.,
2006). It was hypothesized that Si would migrate from non-stressed tissue to salt-
sensitive areas thus protecting the plant against salt stress.
The main aim of this work was to profile the effect that Si would have on salt
resistance of barley and wheat at their initial stage of development.

Materials and methods

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We used C3 plant species barley (Hordeum vulgareL.) as a test plant. Barley is a silicon
accumulating plant having an average total silicon content of 1.21% Si. Barley was
grown in artificial organo-mineral soil having the following concentrations: monosilicic
acid 27–29 mg Si/kg soil, determined by water extraction method, and silicon 340-350
mg Si/Kg soil, calculated with acid-extractable (0.1N HCl) method. Soil moisture was
maintained within the range of 20–25%. Day/night temperatures were 22–24°C/17–19°C.
Natural light intensity in the middle of the day was approximately 900-1200 μmol
photons/m2 s. For 3-4 h/day the window of the greenhouse was opened to let UV light
reach the plants. Barley was grown during a 4 week-period. The investigated plants were
cut near the root level and put into taps with water or 550 mM NaCl solution or kept
without water. The samples of plant tissue (leaf blades, internodes of stems) were
collected just after cutting and at intervals of 24 hours and 48 hours after cutting. The
content of monosilicic acid and polysilicic acid were determined in the fresh plant tissue
and in the apoplast and symplast and total plant Si was analyzed as well. Roots and nodes
of stems were also analyzed for these parameters immediately after cutting.

(). The total Si was determined by Elliot and Snyder (1991) method. Fresh plant tissue
was washed and dried at 65oC. Then the samples were grounded and sieved through a 0.2
mm sieve. The dry sample (about 0.1-0.2 g) was placed into a temperature-resistant 100-
ml plastic tube; after adding 4 ml of 50% NaOH the tubes were incubated for 4 h at room
temperature. After incubation, 2 ml of H2O2 were added to each sample. After the
cessation of gas release, the samples were put into an oven at 100 oC for 3-4 h. The tubes
were removed and allowed to cool before adding double-distilled water to bring the final
volume to 50 ml/tube. The resultant solution was analyzed for Si by colorimetric method
with molybdenum-ammonium technique (Iler, 1979). The method is based on the ability
of Si-anion to form yellow complex with molybdenum with the subsequent formation of
blue complex as a result of reduction by ascorbic acid to increase sensitivity. Strong
H2SO4 and oxalic acid were added to the final solution to decompose the phosphate-
molybdenum complexes and to eliminate phosphorus interference.
Two methods were used for measuring monosilicic and polysilicic acids in different
parts of fresh plants (Matichenkov et al., 2008). First, fresh plant samples (0.1–0.2 g)

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were grounded in a mortar containing distilled water, and the resultant homogenate was
centrifuged for 15 min at 6000 g to precipitate colloids and solid particles. In a part of the
supernatant, monosilicic acid was determined as described above (Matichenkov et al.,
1997). The concentration of polysilicic acid was calculated as the difference between the
concentration of monosilicic acid after de-polymerization of polysilicic acid in the
remaining supernatant and the concentration of monosilicic acid in the first part of
supernatant (Matichenkov et al., 1997).
To determine mono- and polysilicic acids in the apoplast and symplast of plant leaves
and stems, fresh specimens were cut into fragments 2.0– 2.5 cm long, put into a flask
containing distilled water at a ratio of 1 : 100 by weight, and shaken. The apoplast
content of the acids was measured in a series of 1- to 5-ml aliquots, sampled 0, 1, 5, 15,
30, 60, 180, 360, and 1440 min later. The samples of plant tissues obtained after filtering
the remaining solution were homogenized in a mortar and again mixed with the filtrate.
After that, the suspension was shaken for 60 min, and the amounts of mono- and
polysilicic acids were measured once more. The symplast content of mobile forms of Si
was calculated as the difference between the amounts of silicon in the solution after
complete homogenization of plant samples and the amount of Si in the apoplast. The
concentration of soluble Si compounds was determined only for control plants and plants
which were in salt solution during 48 hours.
All treatments were conducted in 5 replications. Each sample of plant tissue was
analyzed by times. Data processing was conducted with the standard programs of
mathematical statistic.

Obtained result and discussion

The obtained results are shown in Tables 1 and 2. The concentration of monosilicic acid
in the sap of the plant tissue ranged from 195.2 to 368.2 mg Si/kg of fresh weight of the
plant. The maximum concentrations were found in the roots and the minimum
concentrations were in the internodes of stems (Table 1). The concentration of
monosilicic acid in the plant tissues ranged from 523.7 to 215.5 Si ppm. The
concentration of polysilicic acid ranged from 363 to 682 mg/kg of fresh weight of the
plant. The maximum concentrations of polysilicic acid were examined in the roots and

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the minimum ones in the noodles of stems (Table 1). The concentrations of polysilicic
acid in the plant ranged from 439.4 to 970.1 Si ppm and this confirms that barley plant
tissue is very high in soluble Si. Considering that soil solution had 27–29 mg Si/kg as
monosilicic acid, our data demonstrated that plants have a mechanism of active uptake of
monosilicic acid against concentration gradient.
Plants that after cutting were kept in the air, showed increased concentrations of
both mono- and polysilicic acids in their sap (Table 1). This may be the result of water
evaporation process. Placing plants in water initiated a decrease of monosilicic acid
content in leaves and of both mono- and polysilicic acids in the stems. We suggest that
this phenomenon is related to the migration of soluble Si into water, however at the same
time, the concentration of polysilicic acid in leaves, increased.
Plants that stayed in salt-bearing solution, have shown a different dynamic in
mono- and polysilicic acids. In leaves, monosilicic acid sharply decreased while
polysilicic acid increased during the first 24 h. During the next 24 h, polysilicic acid
started to decrease. In the stem sap, the concentration of both monosilicic and polysilicic
acids increased (Table 1). We suppose that such process occurs from simulation of stress
conditions and because plant tissues under maximum stress require accumulation of
soluble Si compounds. For treatments under dry condition and experiments with water
the maximum stress is located in the leaves. Salt stress exerts the most influence on the
stem. In fact, salt present in the solution inhibits the movement of soluble Si compounds
from the sap to the solution.
The concentrations of monosilicic and polysilicic acids in the apoplast and
symplast of barley tissue are presented in Table 2. The total concentrations of mono- and
polysilicic acids in the symplast and apoplast of leaves and stems were lower than those
in the sap (Table 1). This is probably related to adsorption of soluble Si substances
during separation of apoplast and symplast.
The simulation of salt stress resulted in a decrease in both forms of soluble Si in
the leaves and their increase in the stems (Table 2). In stems, the maximum increase was
tested for polysilicic acid. This data supports our hypothesis that Si movements in plants,
is realized by polysilicic acid (Biel et al., 2008).

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 Table 1. Soluble Si compounds in barley.
Plant tissue Fresh plant, mg Si/kg Sap, ppm Si Total Si %, % of soluble Moisture of
Monosilicic Polysilicic Monosilicic Polysilicic dry weight forms of Si plant tissue, %
acid acid acid acid
Control plant
Leaf blade 207.6 515 229.3 569.0 1.45 52.4 90.5
Internodes 189.0 525 215.5 598.6 0.91 63.7 87.7
Noodle 195.2 363 236.3 439.4 0.47 68.2 82.6
root 368.2 682 523.7 970.1 3.91 9.0 70.3
Water, 24 h
Leaf blade 147.3 842 165.8 948.2 1.55 56.9 88.8
Internodes 154.7 374 181.5 438.9 0.7 51.0 85.2
Water, 48 h
Leaf blade 114.3 887 132.4 1027.8 1.65 44.3 86.3
Internodes 122.3 265 141.9 307.1 0.51 55.4 86.3
Salt solution, 24 h
Leaf blade 75.56 965 90.9 1161.2 1.23 50.1 83.1
Internodes 218.4 655 250.4 751.1 0.99 68.9 87.2
Salt solution, 48 h
Leaf blade 74.8 402 89.5 481.4 1.05 27.5 83.5
Internodes 158.4 797 189.9 955.6 1.14 50.5 83.4
Dry, 24 h
Leaf blade 234.8 887 294.2 1111.5 1.46 38.0 79.8
Internodes 215.6 589 270.1 738.3 0.48 83.0 79.8
Dry, 48 h
Leaf blade 305.5 980 528.5 1695.5 1.45 21.0 57.8
Internodes 224.3 722 315.4 1015.8 0.47 69.7 71.1
LSD05 5.8 20 5.8 20 0.03 - 0.4

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 Table 2 Mono- and polysilicic acids in leaves and stems of barley.
Monosilicic acid Polysilicic acid
Part of the Apoplast Symplast Apoplast Symplast
plant Si, mg/kg fresh mass
Control plants
Leaf 29 143 319 402
Stem 12 96 88 329
Salt solution, 48 hours
Leaf 21 69 216 302
Stem 26 122 344 585
*LSD05 5 10 10 15

The above data demonstrates that plants contain very high concentrations of
soluble forms of Si. Significant amounts of Si in plant tissue are soluble Si compounds,
which can move to plant’s cells that are under stresses. The elaborated methods make it
possible to obtain new information about the concentration of soluble silicon compounds
in plant sap. Separate analysis of mono- and polysilicic acids in the sap and in the
apoplast and symplast provide knowledge of the biochemical processes that are
controlled by active forms of Si. Salt stress resulted in increased amounts of soluble
forms of Si in plant tissue and this was made possible by the active transferring of Si.

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