The relationship between environmental pollution and nutrition in particular, which forms the basis of health, is fundamentally important for protecting human health. Therefore, the data obtained from the examination of how plants and animals consumed as food are affected by environmental pollution can be seen as an indicator of their effects on humans. On the other hand, the role of technology and nanotechnology in life has been increasing in this century, and a considerable amount of heavy metals, nanoparticles (NPs), and nanotubes (NTs) are released to the environment. The results of morphological or anatomical examination of runner bean (Phaseolus coccineus L) and artichoke (Cynara scolymus L.) plants subjected to copper (Cu) and lead (Pb) heavy metals and chickpea (Cicer arietinum L) plants subjected to Au nanoparticles and C70 single-walled carbon nanotubes (SWNTs) are presented with this study in the point of their phenological development process. The three taxa belonging to Fabaceae and Asteraceae families with high economic status and having flowers with characteristic features were chosen deliberately as representatives. This chapter presents a study that will shed light on future biomonitoring-based studies focusing on the impact of environmental pollution on plants phenology with economic value.
- heavy metal
- runner bean (Phaseolus coccineus L)
- artichoke (Cynara scolymus L)
- chickpea (Cicer arietinum L)
It is a known fact that environmental pollution constitutes an important problem in Turkey as well as in the rest of the world. Rapid industrialization and population growth have caused pollution in the atmosphere, pedosphere, and hydrosphere. Therefore, it is seen that countries pay particular attention to pollution-related studies and health problems caused by pollution and allocate high amounts of resources to deal with the problem.
Heavy metals show toxic effects at certain concentrations for living organisms. However, low concentrations of some heavy metals are essential for normal and healthy plant growth. Furthermore, heavy metals and nanoparticles are causes of concern because they can penetrate into different parts and cells of plants at different rates, and by this way, they enter the food chain and reach the living beings.
There are about 22,000 bryophyte species and 20,000 algae species; however, vascular plants are the dominant plant group in the world with 255,000 species. Land plants, which perform their life cycles completely in the terrestrial environment, are mainly composed of bryophytes and vascular plants. Furthermore, at least a thin film of water is required for fertilization in all taxa except seed plants. Even in the two primitive genera seed plants, cycad and gingko, fertilization is a result of free-swimming spermatozoids released into the liquid medium in the archegonium chamber [1, 2, 3].
One of the most important features of vascular plants is the presence of buds at the ends of the trunk and side branches in the gymnosperms and generally in the angiosperms. The bud is an apical meristem coated with protective bud scales. Meristem is the region of cells to which new cells, tissues, and organs are added and has the potential for active cell division and contributes to plant growth. Therefore, despite the limited growth potential in animals, plant growth is limitless due to the presence of apical meristem. However, the development of plant parts, such as leaves, flowers, and fruits, is limited to their shapes and is genetically predetermined . In short, when evaluated from a phenological point of view, plant parts do not show any further growth independent of the time they remain on the plant after completing their development.
Cell development and differentiation take place as the changes occurring in protoplast; for example with the fusion occurring in vacuoles to grow, via structures such as mitochondria, plastids and the golgi body, endoplasmic reticulum, microtubules, and microfilaments in cytoplasm. Cell walls differentiate and increase in thickness due to structural and environmental effects, and they may become permeable. Moreover, the walls may integrate with the lignin, which increases tensile forces. Tissues formed by the differentiation of apical meristem include parenchyma, collenchyma, sclerenchyma, and primary xylem and primary phloem, in which the pith and cortex are formed [1, 2].
Phenological stages are divided into eight possible principal stages:  bud development,  leaf development,  shoot/branch development,  inflorescence emergence,  flowering,  fruit development,  fruit maturity, and  senescence and the beginning of dormancy . Secondary parts and secondary metabolites occur in the plant during the phenological cycle . Genotype and environmental factors are involved in the emergence of seconder metabolites. In this case, based on the amount of soil, water, and air pollution in the environment in which the plant grows, various deteriorations may occur as a result of morphological and physiological changes whose effects on the plant can be seen with the naked eye or observed only through microscopic examinations. In this chapter, general information about heavy metal and nanoparticles is given, and the effects of heavy metals and nanoparticles on the seedlings of runner bean (
2. Effects of heavy metals, nanoparticles, and nanotubes on plant phenological development
The term heavy metal has been used by scientists with various definitions for about 60 years. An element with a density of more than 7 g/cm3, in 1987 with a density of more than 4 g/cm3, in 1992 with a density greater than 5 g/cm3, and in 1995 with a density of 6 g/cm3 with a metallic property was classified as heavy metal in 1964. Some scientists have classified heavy metals according to their atomic weights, atomic numbers, other chemical properties, and toxic properties. In biological terms, the term heavy metal is generally used for possible contamination of metals and metalloids on the environment and in terms of their toxicity or ecotoxicity .
Heavy metals are released into the atmosphere, pedosphere, and hydrosphere every day due to human activities besides natural causes, such as volcanic activities. Flying ashes from the chimneys of cement plants and thermal power plants; the use of heavy metal paint; the smoke emitted by motor vehicles as well as their plastic-based parts such as brake pads, garbage, and waste sludge incineration plants; and the release of industrial wastes, such as pesticides, fertilizers, paper, batteries, products, etc. are among the main causes of heavy metal pollution [5, 6, 7].
The discharge of heavy metal-containing particles released from the factory and plant chimneys onto agricultural lands, their dissolution in the soil by rain or irrigation, or the irrigation of agricultural land mixed with industrial wastewater leads to various diseases in crops grown on such lands and damages the agricultural economy [8, 9, 10].
Heavy metals have toxic effects for living organisms at certain concentrations. However, certain critical concentrations of some heavy metals are necessary for normal and healthy plant growth. Therefore, heavy metals are classified as essential elements and nonessential elements according to their participation in life processes. Cobalt (Co), copper (Cu), manganese (Mn), molybdenum (Mo), iron (Fe), nickel (Ni), and zinc (Zn) are heavy metals necessary for the growth and vitality of plants and are considered essential elements. Heavy metals such as barium (Ba), cadmium (Cd), mercury (Hg), antimony (Sb), lead (Pb), and chromium (Cr) are not essential for plants and other living organisms and are called nonessential elements .
Essential elements are found as a cofactor in many enzyme systems and as a structural component in biological processes in living organisms. For example, copper is an essential element for normal plant growth at certain concentrations. Copper is an essential cofactor for many metalloproteins in plants and plays a role in photosynthetic electron transport, mitochondrial respiration, cell wall metabolisms, and hormone signal transduction pathways [12, 13].
High concentrations of copper (depending on plant species) show toxicity in plants although it is an essential element. Lead is not an essential element and shows toxic properties for plants. The presence of excess copper and lead in the environment negatively affects phenological development in plants .
These heavy metals result in lipid peroxidation , degradation of cell and thylakoid membrane structure, and a decrease in chlorophyll amount due to the change in the chloroplast structure and thus chlorosis as a result of the oxidative damage they caused . Heavy metals bind to sulfhydryl (-SH) groups of proteins and inhibit enzyme activity  and cause oxidative DNA damage [18, 19], chromosomal abnormalities , and lack of other essential elements [21, 22, 23, 24].
More than 30 base lesions were characterized by DNA exposure to reactive oxygen species . On the DNA, reactive oxygen species can cause single-nucleobase lesions, single-strand breaks, double-strand breaks, and various oxidative damages such as base connections in the strand [26, 27, 28].
Contamination of soils with heavy metals and the accumulation of heavy metals in high concentrations in plants grown here have a genotoxic effect in plants and lead to mutation-like changes in the DNA profile. Therefore, a connection can be established between these changes in the organism and the intensity of pollution in the soil .
Sresty and Rao (1999) examined the ultrastructural changes in the nucleolus, nucleus, endoplasmic reticulum, and vacuoles in pea plant stem cells in response to zinc and nickel stress .
Zengin and Munzuroğlu (2004) observed the root, stem, and leaf growth in bean (
Soudek et al. (2010) exposed flax (
Öztürk Çalı and Candan have studied the effects of fungicide on the morphology and viability of pollens of tomato (
Candan and Öztürk Çalı (2015) have observed pollen micromorphology of four taxa of
Some studies have used various methods based on single-cell gel electrophoresis (comet assay), micronucleus analysis, or cytogenetic analysis in order to investigate the genotoxic effects of pollution on plants.
Steinkellner et al. (1999) treated samples of
Menke et al. exposed the root area of
Today, especially due to increasing demand and changing climatic conditions, many studies have been carried out in plant biotechnology regarding more resistant agricultural plants against factors such as drought, salinity, freezing, and heavy metal contamination. Various biological, chemical, and physical methods are available as regards obtaining biomolecules; for example, nanomaterials with their considerable reactions have much attention in biomass. The basis of this study is to determine the genotoxic effect levels of different stress factors on different plant species. Until recently, the investigation of the effects of stress factors as heavy metals, nanoparticules, and nanoparticles on plant phenology remained at cellular, morphological, and anatomical levels.
3. Materials and methods
Fabaceae family to which runner bean and chickpea plants belong and Asteraceae family to which artichoke belongs were selected as the study material. Both of them are families with large numbers of economically important plants in Turkey as well as in the rest of the world. The flowers of the Fabaceae family are zygomorphic in shape and have legume and lomentum fruit. There are many flowers lined up on the flower tray (receptaculum) and head (capitulum) formed by the bracts surrounding these flowers, and there are achene type fruits in the Asteraceae family [43, 44, 45, 46].
The aim of the present study was to investigate the effects of copper and lead heavy metals on runner bean (
3.1 Germination and cultivation of runner bean and artichoke seeds
Runner bean and artichoke seeds were sterilized and then planted for growing. At least 20 seeds were included and observed in the control group and for each heavy metal application. Seeds were planted and germinated in viols with fine-grained perlite .
CuCl2 2H2O and Pb (CH3COO)2 3H2O solutions were applied in concentrations of 20, 40, 80, 160, 240, 320, 640, and 1280 ppm to the runner bean and artichoke seeds planted in groups of 3(Cu 20, Cu 40, Cu 80, Cu 160, Cu 320, Cu 640, Cu 1280 and Pb 20, Pb 40, Pb 80, Pb 160, Pb 320, Pb 640, Pb 1280). This procedure was repeated for 21 days. The seeds of the control group were planted and irrigated with distilled water. As a result, seedlings of the control group and those subjected to Cu-Pb heavy metal stress were obtained after 21 days  (Figures 7,8).
3.2 Germination and cultivation of chickpea seeds
Twenty chickpea seeds were exposed to 4 ml Au NPs and C70 SWNTs and 15 ml deionized water mixture for 2 days, and they were grown in pots with perlite for 21 days in two groups. The control group of 20 seeds was also grown in other pots with perlite. All the plants in this group were watered every day in the morning only with water. Au NPs and C70 single-walled carbon nanotubes exposed to chickpea plants and control group were taken from the pots after 3 weeks, and herbarium materials were made. On the other hand, some of them were stored at 70% alcohol for microscopical investigations  (Figure 9).
3.3 Methods used to obtain anatomical data
Cross sections were taken from taxa to determine and compare the characteristics of root and stem anatomy of runner bean and artichoke seedlings exposed to Cu and Pb heavy metal concentrations. While determining the samples from seedlings to take sections, 3–10 mm from the end of the roots and the middle part of the body above the ground were used. These fragments were used for sectioning with microtome by the paraffin method. All plant samples were subjected to various treatments to make the sections suitable for microtome removal .
These operations were carried out on the samples taken from the abovementioned parts of the plants retained in 70% alcohol. These parts were passed through 80, 90, and 100% alcohol and 2 alcohol/1 xylol, 1 xylol/1 alcohol, 1 alcohol/2 xylol, and 100% xylol solutions, in this order. The paraffin was allowed to penetrate the interior of the samples which were kept in the laboratory drying oven at 60° for 48 hours. Sections of 20, 25, 30, 35, and 40 μm used in the investigations were obtained via samples placed in paraffin blocks. The sections were placed on slides properly by using a hot water bath set at 40 °, and they were fixed onto the slides with adhesive. Sections were cleared off paraffin using 100% xylol, 1 xylol/1 alcohol, absolute alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and purified water, in this order, for 5 minutes each, and then they were stained with safranin and fast green. Samples were kept in pure water, 70% alcohol, 80% alcohol, 95% alcohol, absolute alcohol, 1 xylol: 1 alcohol, and 100% xylol, for 1 minute each, so that water removal from the tissues was completed [49, 50, 51]. After removal of all the water, the preparations which were made permanent using Entellan and allowed to dry for 4–5 days at room temperature were examined in general. In this way, the reaction of the samples (with different concentrations of heavy metal in the cells) to dyes and the possible staining status were determined.
During the examination of the sections, the treatment of plant tissues with dye has caused a problem since the samples contain heavy metals, such as copper and lead, and they affect the physiology of the plant. However, it is known that in permanent preparations, an artificial appearance is obtained by losing some of the chemical content of the plant material and pigments due to the fact that the plant materials pass through a considerable amount of chemical stages. Therefore, it has been stated in some studies that permanent preparation methods are not suitable for some plants [52, 53]. It was also tried to make hand cross sections on artichoke and runner bean samples. The anatomical features of the taxa were evaluated and interpreted according to Carlquist, Fahn, and Yentür [2, 54, 55].
Dyes were prepared using different ratios of safranin and fast green, and all of them were tested for staining of heavy metal-treated anatomical specimens in accordance with the literature [50, 51], and examinations were performed on the specimens deemed appropriate.
Furthermore, objectives of 4, 10, 20, and 40 were used for examination and photographing the anatomical structures of the root and stem of the taxa. Runner bean and artichoke root and stem photographs were taken with 4, 10, and 20 objectives, and the unit of measurement was determined as 100 (μm).
4.1 Morphological observations
Runner bean seeds were germinated at all concentrations of Cu and Pb. However, there wasn’t any germination observed in 640 and 1280 ppm concentrations of Cu in the artichoke seeds, but germination occurred in all concentrations of Pb [39, 47].
It was determined in the morphological observations of runner bean and artichoke plants subjected to heavy metals that significant differences occurred in different doses of phytotoxic effects of Cu and Pb .
The control group samples of the runner bean and artichoke plants were photographed in order to compare the heavy metal phytotoxic effects on the morphological characteristics of the plants. Moreover, the general appearance and close-up photographs of the samples related to runner bean plant irrigated with Cu 20 ppm and Pb 160 and Pb 1280 ppm concentrations and artichoke plant irrigated with Cu 20 and 40 ppm and Pb 20 and 40 ppm were taken (Figures 10–15).
Cu and Pb heavy metals caused various phytotoxic effects in cases where the recommended dosage was exceeded or excessive pollution occurred in any other way was determined as the result of the study when the changes in the morphological structures of the plants were examined. Phytotoxicity seen in the morphological structure of the plant emerged as bending, shrinkage, and dark spots on the end of the leaves. On the other hand, while plant root, stem, and leaf lengths increase in low doses, high concentrations (640–280 ppm) cause size reduction and incomplete development  (Figures 10–15).
It was observed that there was an increase in plant root, stem, and leaf sizes in both treatment groups when chickpea plant control group and the plants subjected to Au Nps and C70 SWNTs were compared. Furthermore, it was determined that there were increases in the number of fibrous roots, nodes, and subbranches in both groups (Figure 16).
4.2 Anatomical observations
Micrometer was selected as the unit for measurements taken from root and stem cross sections of the runner bean and artichoke seedlings. Cross sections taken from the roots and stem parts of the plants were considered suitable for evaluation. The roots and stems, epidermis, vascular bundle elements, secretary canals, sclerenchyma, starch sheath, cortex and pith cells, and cambium cells were measured, and the presence and variety of crystals were examined and compared .
Photographs of root and stem cross sections of the runner bean and artichoke plants were taken in order to compare the anatomical effects of heavy metal phytotoxic effects on the morphological characteristics of the plants. Runner bean root cross-sectional photographs were taken from root samples subjected to Cu 80 ppm and 640 ppm, and Pb 640 ppm concentrations and stem cross-section photographs were taken from the samples subjected to Cu 20, 80, and 640 ppm concentrations. Artichoke seedlings of root cross sections treated with Cu 160 ppm and Pb 320 and 640 ppm concentrations and stem cross sections treated with Cu 20 and 160 ppm and Pb 1280 ppm concentrations were examined  (Figures 17–25).
However, diseases caused by heavy metal stress in the plant, such as chlorosis and necrosis, and epidermal thickening, density of crystallization, increase in hairiness, and thinning in vascular bundles had negative effects on staining in anatomical studies and caused the tissues not to absorb the dye. Furthermore, the presence of heavy metals in the plant content and crystallization prevented the retention of the dye and made staining process difficult. Thus, a large number of experiments with different dyes and dye concentrations have been carried out for the tissue to absorb the dye into the cell .
Heavy metal pollution in soil and water is one of the most important environmental problems in industrialized countries. Various heavy metals such as cadmium, lead, copper, mercury, and chromium from various industrial establishments such as leather, paint, fertilizer, textile, cement, and chemical industry are released onto soil and aquatic environments and cause environmental pollution [56, 57, 58]. Since most of the heavy metals do not undergo biodegradation in the environment, they can easily accumulate and increase their toxic effects on living things by forming very complex structures .
High-structured plants are equipped with advanced features that allow them to adapt to changes in nature, one of which is the retention of metals in the roots . Retention and deposition of metals in roots have more negative effect on the root area and seed germination than stem and leaf growth. Zengin and Munzuroğlu (2004) reported that the most negative effect was in the root area of the bean (
Soudek et al. (2010) treated linen (
The defense mechanisms developed by plants against heavy metal stress may vary in the level of family, genus, species, subspecies, and variety [54, 55, 56, 57]. The defense mechanisms that allow plants to be tolerant to heavy metals have not yet been fully understood. However, the mechanisms of tolerance include vacuolar phenomena , enzymatic and nonenzymatic antioxidant systems [59, 60], metal-binding ligands such as metallothionein , and alternative oxidase pathways .
Although copper is an essential element, it is more toxic when it is present in high doses compared to cadmium, a nonessential element. This is explained by the direct influence of copper on the formation of reactive oxygen species [superoxide radical (O2˙−), hydrogen peroxide (H2O2), hydroxyl radical (OH−)] because it is a trace element. Since copper and iron transition metals are involved in oxidoreduction reactions, they act as catalysts that accelerate the formation of reactive oxygen species [63, 64, 65, 66]. As in previous studies, it was found that artichoke seedlings are negatively affected mostly by copper in molecular terms [39, 41, 67, 68]. The highest negative effect was observed in groups subjected to copper solutions in terms of root length, root dry weight, total soluble protein amount, and genomic mold stability [39, 40, 41, 42] (Figures 17–22).
Reactions to heavy metal stress in this study emerged as different anatomical results in both plants. However, it can be determined that the response of copper-treated samples was not always greater than that of the lead-treated samples even though they responded differently to different concentrations in terms of anatomical results. Previously the effect rate observed at the molecular level in Cu-treated samples has been found to be higher than the effect rate in Pb-treated samples in both plants [39, 40, 41, 42]. However, it was not the case for the present anatomical study because the roots and stems of the plants were examined in terms of many parameters, so heavy metals did not show the same effect  (Figures 17–25).
It was observed that seedlings belonging to runner bean species showed high tolerance against lead and copper stress. The genome of the plants was preserved at 94–95%, and no significant reduction in total soluble protein was observed especially at 1280 ppm concentration of lead and copper solutions. This situation has led to the conclusion that runner bean species has a strong defense mechanism against heavy metal contamination [40, 41, 42, 49]. In this anatomically based study, it would be wrong to say that one heavy metal is always superior to another in terms of its effects because plant parts were examined anatomically in terms of many parameters and similar results were not observed in all. The reaction of the plants as crystallization is particularly important and has shown similar responses in both heavy metal treatments. The differences varied in terms of crystal density, location, and shapes (Figures 17–25) .
As a result, various anatomical differences determined in this study regarding the characteristic features, such as root and stem vascular bundles thicknesses, cell size and fragmentation in the pith region, formation or thickening of cambium and sclerenchyma, shape and size of secretary canals, differences in cortex cell sizes, the sizes of the vascular bundles, the formation of crystals and deposits in the phloem layer according to the heavy metal concentration applied, the number of epidermis cells per unit area, and the epidermal wall shapes, can be used to reveal the phytotoxic effects of Cu and Pb heavy metals.
The morphology of roots and shoots is extremely important for the growth and development of all plants, and each factor that changes their morphology has positive/negative effects .
On the other hand, Candan and Lu (2017) have shown that there are more differences on the pea green (
It is the fact that the application of some materials which are not used consciously or at the recommended dosage and which contain heavy metals in order for the crop to be attractive for the consumers actually yields negative results. Therefore, this study is important about examining the extent of heavy metal phytotoxic effects related determining them on plants phenological development in the point of morphologically and anatomically changes. The information given in this study is valuable as it presents the negative molecular effect of heavy metal pollution on the plant in terms of morphological and anatomical aspects. Furthermore, this study will guide the researchers on the effects of environmental pollution in relation with the phenological development of economic plants and hence on human health.
Heavy metal and nanoparticule-nanotube-induced plants must be evaluated in the point of biochemistry and examined via scanning electron microscope (SEM) and transmission electron microscope (TEM) regarding their root, stem, and leaf structure and apical tip, leaf-bud primordiums, and provascular tissue in detailed ways for interdisciplinary studies according to plants’ phenological development progress.
I would like to thank Manisa Celal Bayar University (Turkey) Scientific Research Projects Coordination Unit (BAP) for supporting most of this study as part of the projects numbered 2012-057 and 2014-120.
I am also thankful to Dr. Qi Lu, a member of the Department of Physics and Engineering, and Dr. Gulnihal Ozbay, a member of the Department of Agriculture and Natural Resources at Delaware State University (USA), for the support of some laboratory studies related to nanoparticles and nanotubes.
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