Research objects were leaves of three hydrophytes: Myriophyllum spicatum (Haloraceae), Potamogeton pectinatus (Potamogetonaceae) and P. perfoliatus (Potamogetonaceae), which were identificated according to Manual on High Plants of Ukraine [17] . The leaves of plants were collected at the beginning of June at the stage of vegetative growth. We used upper leaves that finished growth by enlargement. Plants were grown in water along-shore of the Rusanivskiy canal (left Shore of Dnipro River, in Kyiv) on the depth of 80 - 100 cm. The sun illumination [photosynthetic photon fluency rate (PPFR)] on water surface was 1200 - 1250 µmol quantum∙m⁻²∙sec⁻¹, and on surface of the upper leaves of studied plants (about 8 - 10 cetimetres below the water surface) was 12 - 15 µmol quantum∙m⁻²∙sec⁻¹. PPFR was measure by the means of Light Meter Li-250 (USA, LI-COR). The leaves situated above 8 - 10 cm under water surface were used for cytochemical analysis. The temperature of water was +15˚C and the temperature of air was +23˚C. The leaves of three plants from each species: submerged dissected leaves of M. spicatum (Figure 1(a)); submerged entire leaves of P. perfoliatus (Figure 1(b)), and submerged dissected leaves of P. pectinatus (Figure 1(c)) have been used for the cytological investigations.

The information concerning the physic-chemical property of water in Dnipro river is given by the Management of Dniepr-water resources (2015, http://www.dbuwr.com.ua/sendmail.php) and the data of ecologists [18] . Water of Dnipro River is the second class. Salts composition of water in Dnipro River (near Kiev) are carried out over last several years shown that water is clean and alkalescent, characterized by the middle salts value, notable: mineralization (sum of ions) average from 250 to 500 mg/dm³; chlorides―16 - 24 mg/dm³; sulphates― 13 - 53 mg/dm³; ammonium ions―0.29 - 1.08 mg/dm³; and dissolved oxygen―8.7 - 13.5 mg/dm³ (Management of Dniepr-water resources, 2015, http://www.dbuwr.com.ua/sendmail.php; [18] ).

The cytochemical method accordingly to [19] was used for the study of both distribution monolignols (syringyl and guaiacyl) and relative content of these monolignols in walls. Leaf sections were hand cut from fresh leaf samples. For the detection of phenylpropanoids, sections were stained within 2 min with saturated (0.25%, w/v) diphenylboric acid-2-aminoethyl ester (DPBA) (Sigma) in MilliQ water containing 0.02% (v/v) Triton-X-100, then samples intensive washed with H₂O. The sections were visualized immediately with an laser scanning microscope LSM 5 Pascal (Carl Zeiss, Germany). For detection of complex DPBA-syringyl fluorescence a laser was excited at 340 - 380 nm, and the fluorescence emission detected at 430 nm wave length; and for detection of complex DPBA-guaiacyl fluorescence a laser was excited at 450 - 490 nm, and the fluorescence emission detected at 520 nm wave length, using an ×10, ×20 and ×40 objectives the PASCAL program. Chlorophyll auto fluorescence was excited at 440 nm and fluorescent emission detected at 660 nm. Fluorescence intensity of monolignols was measured in the cell walls as a function of emissions wave length using the PASCAL program.

For statistical treatment took for two/three leaves from each plant, in every leaf took at least 30 different cells (epidermis, photosynthesizing parenchyma, and xylem vessels). Values of cytochemical results were expressed as the mean and standard errors. Three replications of cytological results were expressed as the mean and standard arrows. Statistical significance of relative content of monolignols in cell wall was determined using the BIO software (Institute of Botany, Kiev, Ukraine) and a Student’s test (p < 0.05).

Cytochemical analysis of monolignols in the dissected leaves of M. spicatum are shown as blue fluorescence for syringyl and as green fluorescence for guaiacyl in the walls of epidermis, photosynthesizing parenchyma and also in xylem vessels (Figures 2(a)-(d)). But the fluorescence intensity of DPKK-syringyl and DPKK-guaiacyl complex was different in the tissues (Table 1; Figure 2(e) and Figure 2(f)). Wall of vessels and anticlinal wall of epidermis had the greatest fluorescence intensity of DPKK-syringyl and DPKK-guaiacyl complex in comparison with other walls of epidermis and parenchyma (1^st - 4^th layers). It should be noted that in the corners of parenchyma cells fluorescence of monolignols was considerably higher, than along walls. Except it, attitude of content of syringyl toward guaiacyl in the walls of photosynthesizing parenchyma was high enough, especially in the first layers to the parenchyma. The size of S/G ratio in cells takes place in the next order: anticlinal walls of epidermis > walls in the corners of parenchyma > vessel walls > parenchyma walls > periclinal walls of epidermis. It is revealed that maximum frequency for syringyl in the epidermal cell was 225787 (pixels, blue graph), the maximum frequency for guaiacyl was 286463 (pixels, green graph), and the maximum frequency for auto fluorescence of chlorophyll was 358132 (pixels, red graph) (Figure 2(g)).

^*Values of guaiacyl is significantly different (p ≤ 0.05) from syringyl.

Like fluorescence of monolignols observed in P. perfoliatus leaves. Cytochemical analysis of the complex DPKK-syringyl and DPKK-guaiacyl in leaves shown blue fluorescence of syringyl and green fluorescence of guaiacyl in cell walls of epidermis, photosynthesizing parenchyma and vessels cells (Figures 3(a)-(c)). There were some differences in fluorescence intensity of monolignols in cell walls of epidermis, in the corner of parenchyma cells and cell of vessels. The level of fluorescence intensity of monolignols is presented in the Table 2 and Figure 3(d) and Figure 3(e) (diagram). It is necessary to note that syringyl was not exposed in cellular walls of 2^nd, 3^rd and 4^th layers of parenchyma (of three plants) and in plans four-intensity of fluorescent was 7 ± 0.6 relative units; notably, minute intensity. On the size of the relation of S/G cells in P. perfoliatus are situated in such order: walls in the corners of parenchyma > vessels > periclinal walls of epidermis > anticlinal walls of epidermis > parenchyma. Luminescence intensity of monolignols in P. perfoliatus leaves differ from M. spicatum leaves: 2.1 - 3.2 times guaiacyl increased in epidermis walls and 5 times―in walls of 1^st layer of parenchyma. It is necessary to note that independently from leaf species the relative content of monolignols in vessel walls was more than other tissue walls. It is revealed that maximum frequency for syringyl in the epidermal cell was 314166 (pixels, blue graph), the maximum frequency for guaiacyl was 209942 (pixels, green graph), and the maximum frequency for auto fluorescence of chlorophyll was 400695 (pixels, red graph) (Figure 3(f)).

Cytochemical analysis of monolignols in leaves of P. pectinatus is showed blue fluorescence of syringyl and green fluorescence of guaiacyl in the cell wall of epidermis, parenchyma and vessels cells, similar to that in leaves of M. spicatum leaves and P. perfoliatus (Figures 4(a)-(с)). The level of luminescence intensity is presented in Table 3 and Figure 4(d) and Figure 4(e) (diagram). The analysis of monolignols luminescence intensity showed that relative content of guaiacyl in walls of periclinal walls of epidermis, in corners of parenchyma cells and in vessels was more than that in cell walls of anticlinal walls of epidermis and parenchyma cell walls. The size of S/G was lowest in the corners of parenchyma cells. The size of S/G ratio in cells takes place in the next order: parenchyma > epidermis > vessels > walls in the corners of parenchyma. It is revealed that maximum frequency for syringyl in the epidermal cell was 104477 (pixels, blue graph), the maximum frequency for guaiacyl was 24534 (pixels, green graph), and the maximum frequency for auto fluorescence of chlorophyll was 61963 (pixels, red graph) (Figure 4(f)).

Thus, we showed the presence of syringyl and guaiacyl monolignols in submerged leaves of three species real hydrophytes (Myriophyllum spicatum, Potamogeton pectinatus and P. perfoliatus) and also the increased content

^*Values of guaiacyl is significantly different (p ≤ 0.05) from syringyl; ^**In leaf cells of three plants syringyl was not exposed, and in plans four-inten- sity of fluorescent was 7 ± 0.6 relative units; notably, minute intensity.

^*Values of guaiacyl is significantly different (p ≤ 0.05) from syringyl.

of guaiacyl in walls only of two species of Potamogeton. There were the general and differentia signs concerning these monolignols in hydrophytes leaves. General signs were: 1) presence of syringyl and guaiacyl at the inves- tigated species regardless of conditions of growth of leaves; 2) almost identical (sufficiently great) content of S/G

in the vessels cells of all hydrophytes, and in the corners of parenchyma cells of M. spicatum and P. perfoliatus; and 3) certain polarity of S/G ratio, that characteristic for every species. Differentia cytochemical signs were: relative content of syringyl and guaiacyl in different tissues of hydrophytes, and also different quantity of S/G in epidermis, parenchyma and vessels walls of all studied hydrophytes.

In consideration of presence of magnified content of syringyl and guaiacyl in the corners of parenchyma cells of the investigated samples of hydrophytes and the well-known data that the middle lamella and the cell corners are the so-called nucleation sites of lignin and also they rich in Ca²⁺ pectate [8] [20] and are the first sites to be lignified, we can to draw a conclusion that cell corners of parenchyma cells of hydrophytes is also leading spots for lignin synthesis.

The cytochemical analysis of monolignols in different tissues of submerged leaves of M. Spicatum, P. pectinatus and P. perfoliatus) revealed the increase content of syringyl and guaiacyl in epidermis cell walls in comparison with walls of inner layers of photosynthesizing parenchyma. It is known that submerged leaves of hydrophytes are losing trichomes, stomata and thicker cuticle [5] ; therefore the surface of epidermal cells in submerged leaves become more accessible for an invasion of pathogens. Besides, it is known that in cells of leaves and shoots are occurs an increase in lignifications in responses to attack by pathogens (bacteria, fungi) [21] . Besides, it is known that epidermis is the one barrier and protection of leaf from action of ultraviolet, which is provoke both the intensified synthesis of lignin [22] . Taking into account the above and the results of our experiments the greatest fluorescence of monolignols the epidermis cell of leaves that situated under water surface, it is suggested that walls epidermis of studied leaves of hydrophytes protects of leaves from pathogens and even from little UV radiation in water.

Consider that with the increase of quantity of S/G as a chemical barrier increases to protecting of cells from penetration of water and invasion of pathogens [23] . Except it, it is known that this sign (S/G) testifies to the increase of mechanical durability of cells [24] . Taking into account, that the flooded leaves constantly are in a contact with a surrounding water micro flora and numerous alga, and also that flooded leaves undergo permanent pressure of water and action of waves [5] [25] , it is possible to assume that periclinal and anticlinal walls of hydrophytes epidermis protect the surface of cells from exogenous influence of water surroundings therefore occurs defined synthesis of components of lignin. Notably, permanent water surroundings are one of basic exogenous factors of an increase synthesis of syringyl and guaiacyl in the leaves of the investigated hydrophytes. We see in addition, that a presence of these monolignols, and also their ratio in the cell walls of epidermis, parenchyma and vessels correlates with the data that were received of angiosperms dicotyledons upland plants [13] [26] . We do not eliminate, that the leaf cell walls of investigated hydrophytes contains and the third monolignol-p-hydroxyphenyl, that also plays a substantial role in lignin structure, and it is able determine with using other method, UV spectroscopy [27] .

Thus, the obtained results revealed the particularities of syringyl and guaiacyl monolignols of cell walls of Myriophyllum spicatum. Potamogeton pectinatus and P. perfoliatus submerged leaves, which can be used as a basis for future study of both adaptation mechanisms of agricultural plants during flood, and possible for gene- engineering of plant stability in flooded area.