Deposition of Calcium Oxalate Crystals and Tolerance of Deciduous Trees to Pollution

Plants are frequently utilized as biological indicators for the extent of air pollution. They are organisms which are sensitive to pollutants in the air and many studies are focused on morphological, physiological, and histochemical effect of air pollutants on plants. Various experiments have been conducted to explain the interaction of plants and those pollutants. Deciduous tree plant speciesgrowing naturally in Bulgaria, such as Acer campestre L. (field maple), A. tataricum L. (tatarian maple), and four cultivated: A. negundo L . (box elder), A. saccharinum L. (silver sycamore), Morus alba L. (white mulberry), and Platanus acerifolia Willd. (London plane) have been investigated. There were found increasing the formation of Ca-oxalate crystalsin the lamellae of Morus alba L. under pollutedair conditions or scattering of them in different way in Acernegundo L . The most pronounced depositions of different types of calcium crystals were observed in the most resistant to pollution and dry environment species Morus alba L. Further investigations are needed to specify the relation between biomineralization and specific environmental conditions.


Introduction
Calcium oxalate crystals occur in more than 215 higher plant including gymnosperm and angiosperm families (McNair, 1932;Franceschi and Horner, 1980;Lersten and Horner, 2006).The morphologies and precise locations of crystals are under strict genetic control (Franceschi and Nakata, 2005). Therefore, the shape and location of the crystals within a taxon are often very specific and may be represented as a taxonomic character (Genua and Hillson, 1985;Prychid and Rudall, 1999;Lersten and Horner, 2000). Nevertheless, it is found that their shape, size and formation greatly changes under specific environ-mental circumstances (Umemoto and Hozumi, 1972;Weiner and Dove, 2003;Nakata andMcConn, 2007;Tomašević et al., 2008;Gostin I., 2010;Khan and and Siddiqi, 2014;Gostin I., 2016;Cuadra and Cambi, 2017).
Even though the CaOx crystals have intrigued plant scientists for a long time, their functional roles are still poorly understood (Tooulakou et al. 2016). In Tradescantia pallida, crystal formation is influenced by mercury stress that increasedraphide and prismatic crystals in the treated plants (Khan and Siddiqi, 2014). Plantsgrowing under Hg stress have high metabolic rate that increase oxalic acid formation and formation of crystals which may be due to effort of plants to maintain ionic equilibrium (Khan and Siddiqi, 2014). One of the possibilities is that their accumulation is an indication that these crystals are doing effective role in sequestration of metal ions (Franceschi and Nakata, 2005).
There are many evidences that the environmental factors such as air pollution, metal ions, salinity and other, influence on the number, size and shape of formed crystals within the species (Yang et al., 2000;Choi et al., 2001;Gostin., 2010 andGostin., 2016). The addition of the heavy metals to the nutrient medium decreased the number of calcium oxalate crystals of hydroponically grown plants Phaseolus vulgaris (Jáuregui-Zùñiga et al., 2005).Mercuric chloride at high doses increased all types of crystals in Tradescantiapallida as compared with low doses (Khan and Siddiqi, 2014).Cd, Pb, Ni, Mn and Al are detoxified through formation of metal-organic acid or metal-amino acid complexes. Cd and Zn resistance of Atriplex halimus L. is due to precipitation of metals in oxalate crystals (Lutts et al., 2004).In Oryza sativa synthesis of oxalate represents a significant response mechanism that enhances tolerance to Pb (Yang et al., 2000). Crystals of tobacco seedlings are composed of Ca-oxalate, in which toxic Cd is embedded, and under Cd stress production of that crystals increase (Choi et al., 2001).

Material and methods
The material used for this study was collected from two regions: heavily polluted (42°47'N; 23°30'E) and relatively clear (42°30'N; 23°15'E).The trees from the both fields were sun exposure with uniform height and Plant tissue samples were taken randomly, from the middle parts of the leaf blades and fixed in FAA -90% 3 3 ethanol (90 cm ); iceacetic acid (5 cm ); and formalin (5 3 cm ). Standard histological techniques were used to examine the anatomy of the leaf blades.

Results and discussions
In the lamellae cross sections of observed species have been found numerous calcium biomineral formations in the leaf blades of plants from both regions ( figure 1, 2, 3, 4). Among the various plant nutrients, calcium appears to occupy a unique position, acting as an important regulator in many processes related to both growth and responses to environmental stresses (Lautner and Fromm, 2010). Oxalate is a common 2+ cellular constituent involved in Ca regulation, ion balance, and metal detoxification (Franceschi and Nakata, 2005;Rahman and Kawamura, 2011). Ordinarily, plants exposed to stressful air conditions formed in the leaves numerous single or aggregated crystals (Tomašević et al., 2008).
In laminas of Morus alba L. (figure1), formation of Ca oxalate druses and carbonate formations as cystolithswere found much more in leaf blades from polluted region (figure1, A) than those in control samples (figure1, B). The idioblasts of bigger size,cystolith-containingidioblasts were found on the adaxial epidermis of leaves (figure1, A), and in the mesophyll (figure1, B). According to Katsumata (1971), mulberry varieties can beclassified based on the morphologyof cystolith-containingidioblasts. In no other species the idioblasts have not been observed in their cross sections of leaf blades. Removalof heavy metal by accumulation in calcium crystals appears to be common mechanism among plants known to be tolerant to pollution.  Ca-oxalate plays role in biologically controlled mineral deposition (Arnott 1983 andWebb 1999) and it is deposited in intravascular membrane chambers of specialize cells, idioblasts (Webb, 1999). For example, Armeria maritima accumulates Cu in idioblasts, by chelation with phenolic compounds (Lichetenberger and Neumann, 1997). Heavy metals ions are accumulated in calcium-containing crystal in idioblasts (Van Balen et al.1980;Franceshi and Schueren, 1986;Van Stevenick and Fernando, 1995;Mazen and El Maghraby, 1997;Webb, 1999).
The calcium oxalate crystals have been observed on the cross sections of Acer negundo L. from both areas (figure3), but in polluted region the deposition in spongy parenchyma was much higher.The calcium oxalate crystals were observed in Acer negundo L. from Toma, et al., (2015) in the palisade mesophyll. In the clear environment the mineral formations were also mainly in palisade ( figure 3,B).
In the investigated cross sections ofAcer tataricumL. the mineral formation were found in both samples, polluted and control, scattered in palisade and spongy parenchyma (figure4, A, and B). In the tissue, they were densely distributed in the spongy tissue.According to Toma, et al., (2015) in Acer tataricum L. the crystals as rhombic prisms are situatedmainly in the spongy tissue.
In figure 5 it can been seen that in Acer campestre L. calcium crystals are situated in all mesophyll, and

Deposition of Calcium Oxalate Crystals and Tolerance of Deciduous Trees to Pollution
around the vascular bundles (figure5, A and B). There were not pronounced differences in crystal deposition in the samples from both regions. In Acer campestre L.rhombic prism are found in the palisade (Toma, et al., 2015). According to Toma et al.,(2015), the presences of the crystals are most probably influenced by ecological factors.
In the leaf of Platanus acerifolia Willd, druse crystals were observed under palisade in upper parenchyma tissue ( Figure 6). About 9-10 crystals which arranged in one row of cells were observed along the margin between palisade and spongy mesophyll. However, only a few druse crystals were present in the palisade tissue. Additionally, some of the druse crystals were in contact with the xylem or phloem vessels.
The formation of biominerals occurs as a result of interactions between biological activity andexact environmental conditions (Weiner and Dove, 2003).The mechanisms controlling calcium oxalate formation and typeofcrystals in plants remain largely unknown,mostcertainlymustbeacombinationofgenetic s,environmentalconditions,andnutrientavailability (Nakata and McConn,2007). Ingingko leavesgrown in a clean atmosphere crystals were small isolated sparsely scattered throughout the mesophyll, while in leaves from high polluted areas were found in clusterslarger

Snejana Boycheva Dineva
and more irregular crystals lying along prominent veins (Umemoto and Hozumi, 1972).Thealterations of size, type and distribution of calcium crystalswere found among the plants, which aredeveloped on soils with different salinity and amount of calcium (Cuadra and Cambi, 2017).
In Abies alba L.the needles from polluted region contain more calcium oxalate crystals observed as higher density compared to control (Gostin, 2010). Also it was found from Gostin (2016) that in the plants originating from extensively polluted area the frequency of the calcium oxalate crystals is increased. The similar results were found from Tomašević et al., (2008), the leaves of Aesculus hippocastanum L. (Horse chestnut) and Corylus colurna L. (Turkish hazel) exposed to stressful airconditions were with numerous single or aggregated crystals.

Conclusions
In the polluted air environment the changes of leaf blade anatomy have been detected, as well as a deposition of biominerals. The observations showed that in the experiential tree species from the polluted region the formation of crystals in the leaf blades is increased in some of them, while in others have been changed the pattern of scattering.In the light of the collected data and literature survey it can be concluded that there is no absolute and clear correlation between the presence of calcium oxalate crystals and tolerance of the plants to air pollution. Nevertheless, the biomineral depositions are strongly connected with the environmental conditions and can be used as biomarker for the certain environmental factors, but more data should be gathered.