The Effects of Lead on the Meristem of Wheat Seedlings

The ultrastructure of apical meristem cells was studied in Triticum aestivum L. cv. “Trizo” seedlings grown on soil without or enriched with selenium and survived 14 days’ stress caused by lead pollution in the soil. The soil treatments: control—the original soil; (Pb1)—50 mg∙kg −1 ; (Pb2)—100 mg∙kg −1 ; (Pb1 + Se1) —0.4 mg∙kg −1 Se added to Pb1 treated soil; (Pb1 + Se2)—0.8 mg∙kg −1 Se added to Pb1 treated soil; (Pb2 + Se1)—0.4 mg∙kg −1 Se added to Pb2 treated soil; (Pb2 + Se2)—0.8 mg∙kg −1 Se added to Pb2 treated soil were used. Light and other conditions were optimal for plant growth. A distinctive feature of the cells of the apical meristem of control plants was the absence of nuclear membranes. Proplastids were membrane vesicles 1 - 2 microns in diameter, filled with contents of varying degrees of density, from membrane vesicles containing only plastid DNA up to a fully formed structure of proplastids. In (Pb1)-plants, the amount of cytoplasmic ribosomes and proplastids and is not yet surrounded by a plastid membrane.


Introduction
Soil lead contamination caused, depending on the pollutant concentration, inhibition of plant growth, a decrease in the concentration of chlorophyll and photosynthesis, the excessive accumulation of H 2 O 2 and thiobarbituric acid-reactive substances, and a change in the activity of antioxidant enzymes [1] [2].
The introduction of selenium into the soil had a multidirectional effect at different concentrations of the agent [1] [2]. Enrichment of soil with less dose of Se (0.4 mg•kg −1 ) without Pb resulted in: stimulating growth processes; increasing ascorbate peroxidase activity in leaves and the activities of ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase in plant roots; and reducing the amount of thiobarbituric acid-reactive substances. The introduction of the same dose of selenium into soil contaminated with Pb reduced the negative impact of lead on plants. The protective role of Se is considered to be related to decrease of heavy metal uptake and toxicity [3] [4].
Introduction of a larger (0.8 mg•kg −1 ) dose of selenium into lead-free soil caused the suppression of growth processes and an increase in the thiobarbituric acid-reactive substances content during the activation of antioxidant enzymes in the roots and leaves [1]. The introduction of this dose of selenium into soil contaminated with Pb enhanced its negative effect, especially in the variant with a high concentration of lead [2].
As was also shown in the previous article [2], in wheat seedlings growing for 2 weeks in the light in pots with lead contaminated soil, the tissue of leaf blades became almost white. In the mesophyll cells of the whitened leaves, chloroplasts were absent, but there were etioplasts, which in this case were actually proplastids that did not develop into chloroplasts. In other words, despite the growth of the leaf plate, the formation of chloroplasts from proplastids was disrupted. All other cellular structures of leaf mesophyll cells did not differ from those in control plants that were not affected by the action of the stressor. The selectivity of the suppression of the chlorophyll synthesis process and, accordingly, the impossibility of the development of proplastids to chloroplasts indicated a point (target) effect of lead on this process. We assumed that the targets of the action of lead on the development of proplastids to chloroplasts are meristematic cells.
It is known that in the cells of the intercalary meristem, proplastids are well developed and are 1 -2 μm bodies, covered with a double membrane and containing a dense matrix. The cells of the growth cone meristem (apical meristem), in contrast to the intercalary meristem, are characterized by heterogeneity of the proplastid structure [5]. The structure of the proplastids of the apical meristem varies from transparent membrane vesicles containing plastid DNA to fully formed proplastids with a dense matrix (see [5]; and Figures 1-4). Such variability in the structure of proplastids suggests that in the apical meristem there is a process of formation (assembly) of the proplastid bodies [5], and not their simple division, as is commonly believed [6].
To clarify the effects of lead on the formation and development of proplastids in the apical meristem and the possibility of reducing the negative effects of lead at low concentrations of selenium, the aim of this work was to study the ultrastructural organization of the cells of the apical meristem of wheat seedlings survived 14 days' stress caused by lead pollution in soil (50 and 100 mg•kg −1 ) without or with soil enrichment with selenium (0.4 and 0.8 mg•kg −1 ). A 14-day growth period was selected based on previous studies [2] that showed that the leaves of the Pb-treated plants were green during the first week of growth; after another week, etiolation occurred in the middle and bottom parts of these leaves.

Plant Growth Conditions
The seeds of Triticum aestivum L. cv. "Trizo" (species Lutescens) after sterilization by 3% H 2 O 2 for 10 minutes were soaked in distilled water for 24 hours and germinated on moist filter paper in the dark for 2 days. Equally well-germinated seeds were sown in plastic pots (18 seeds per pot). The pots were filled with slightly acidic soil with low available nitrogen; medium contents of phosphorus and selenium, and the higher content of potassium (see [1]

Soil Treatments
Seven variants of the soil treatments in 3 replicates were prepared for plant cul-

Lead Content Measurements
The decomposition of samples of dry plant material (leaves, roots, apical meristem) was carried out by wet ashing with a mixture of nitric acid and potassium nitrate on an electric stove, and then in a muffle furnace with an increase in temperature from 250˚C to 450˚C. The microelement analysis of the samples was carried out by the method of inverse voltammetry on an AKB-07 MK instrument (Akvilon).

Electron Microscopy Analyses of Meristematic Cells
Pieces of shoots adjacent to the caryopsis 2 to 3 mm in height were fixed in a 2%

Lead Content in Leaves, Roots and Apical Meristem
The lead concentration in the leaves of plants was relatively low in all treatments (Table 1). The concentration of lead in the roots and apical meristem was much higher than in the leaves. The introduction of selenium into soil contaminated with lead led to an increase in the absorption of Pb by plants, compared with the variants without selenium, especially to the roots and apical meristem. Characteristically, a lower dose of selenium (Se1) led to greater stimulation of lead uptake by plants, compared with a higher dose of selenium (Se2).        (Table 2). However, the structure of the forming proplastids (Figure 3 proplastids containing only plastid DNA and fully formed proplastids with membrane thylakoids is observed, and the cytoplasmic ribosomes are more numerous than in meristem cells of control plants (Figure 3(b), Table 2). The ultrastructure of meristematic cells of (Pb1 + Se2)-plants was the same as that of (Pb1 + Se1)-plants (data not shown).  Values are expressed as means ± S.D.; the data are estimates and are not intended for statistical accuracy. CellBio

Ultrastructure of Meristematic Cells
In (Pb2)-plants grown with lead content in the soil of 100 mg•kg −1 , signs of degradation are observed in the cells of the apical meristem ( Figure 4). Namely, the density of ribosomes in the cytoplasm decreases to their complete absence in some cells (Figure 4(a), Table 2). Unlike the nuclei of the meristematic cells of control plants that are not covered by the nuclear envelope, the nuclei of the meristematic cells in (Pb2)-plants have a clearly visible envelope (Figure 4(a)). The number of proplastids visible in the cross section is reduced (Table 2), juvenile proplastids are absent, and the formed proplastids have a much smaller diameter (0.5 µm) than in the control (Table 2), and their matrix is compacted ( Figure   4(b)).
With the additional introduction of 0.8 mg•kg −1 selenium into the soil, (Pb2 +  Table 2). The ultrastructure of meristematic cells of (Pb2 + Se1)-plants was the same as that of (Pb2 + Se2)-plants, but signs of degradation of proplastids were less pronounced (data not shown).

Discussion
In previous works of this series of experiments [1] [2], mesophyll chloroplasts of  stressed plants showed signs of degradation. In most mesophyll cells chloroplasts were completely absent, the plastids were represented proplastids which did not develop into chloroplasts. Therefore, we assumed that Pb is likely to target meristem. Antagonistic effect of low concentration of Se and Pb in combination allowed to propose that Se generates some barriers for metal transfer from the wheat roots to the shoots as it was shown in mustard [3] and rice [4].
Surprisingly, it is the lover dose of selenium in the soil (0.4 mg•kg −1 ), useful for wheat plants [2], that stimulates the intake of lead into plants, especially to the roots and apical meristem. We believe that selenium prevents the penetration of lead from the apoplast into the symplast.
On the other hand, the lower lead content in the leaves, roots and apical meristem of (Pb2 + Se2)-plants compared with the (Pb2 + Se1)-plants may be associated with the deep phase of development of extreme stress, suppression of the general level of metabolism. When inhibiting growth processes, the concentra-tion of plastid pigments, the intensity of photosynthesis, and also reducing not only the activity of antioxidant enzymes, but also the intensity of peroxide processes in (Pb2 + Se2)-plants [1], the ability of plants to absorb nutrients from the soil, including the lead can be reduced.
Here we can note also that selenium when applied against a background of lead, compared with lead without selenium, probably caused stimulation of protein synthesis, as evidenced by a sharp increase in the number of ribosomes in the cytoplasm. However, protective inhibition of degradation, for example plastid DNA, was not detected.
Direct division of proplastids in the apical meristem of wheat was not observed. The increase in the number of proplastids required for cell division of the meristem seems to be carried out by a different mechanism (pathway) [5].
As shown in this work, in the cells of the apical meristem, the formation of juve-  Based on the concept [5] about the formation (assembly) of proplastids in the cells of the apical meristem, we believe that toxic agents, such as lead, which inhibit the development of proplastids into chloroplasts in mesophyll cells, act on apical meristem cells at the stage when plastid DNA is replicated in the cytoplasm and is not yet surrounded by a plastid membrane.