Turkish Journal of Botany Turk J Bot (2019) 43: 785-797 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1812-46

Investigating the anatomy of the halophyte Salsola crassa and the impact of industrial wastewater on its vegetative and generative structures

1 1 1 2 Narjes S. MOHAMMADI JAHROMI *, Parissa JONOUBI , Ahmad MAJD , Mansooreh DEHGHANI 1 Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran 2 Research Center for Health Sciences, Department of Environmental Health, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran

Received: 23.12.2018 Accepted/Published Online: 18.06.2019 Final Version: 21.11.2019

Abstract: Salsola crassa M.B. is one of the most successful plants used against industrial pollutants. In this study, we selected young Salsola crassa M.B. plants from their natural habitats. Some plants were irrigated with industrial wastewater containing Fe, Co, Ni, Cu, Pb, and Zn, and others were irrigated with tap water for 3–4 months. Afterwards, the shoots and roots were randomly cut, separated, fixed, dyed, and observed using light microscopy. Structural changes were analyzed by stereology. There were some differences in appearance and structure between the treated and control samples. For example, the number of leaves and flowers and the size of seeds and flowers in the treated plants were reduced. The diameter of the cortical parenchyma, the total area of each vascular bundle, surface area of the pith cells in the stem, leaf cuticle thickness, mechanical layer thickness of the anther, and diameter of pollen grains were reduced. A peculiar palisade chlorenchyma beneath the epidermis and unexpected small vascular bundles on the upper part of the cortex were features of both groups. Finally, we concluded that industrial waste water pollutants affected various aspects of plant development.

Key words: Salsola crassa, resistant halophyte, anatomy, stereology, wastewater treatment

1. Introduction removing N and P from land-based intensive marine Pollutants affect various aspects of plant development, aquaculture farms (Webb et al., 2012). Devi et al. (2016) such as developmental stages, structure, fertility, and seed introduced Suaeda fruticosa L. and Atriplex lentiformis germination. Different plants exhibit various responses (Torr.) as the best salt hyperaccumulator plants. to pollutants in numerous ways. Industrial pollutants, Desert xero-halophyte plants may encounter multiple especially heavy metals, affect distinct features of plants tensions caused by high soil salinity, heavy metals, organic and can even cause death; however, hyperaccumulator pollutants, and long-term water shortages (Toderich plants are the least affected. One of these, Salsola crassa is a et al., 2010). It has been suggested that they can adapt species in the family Amaranthaceae and is a fast-growing to environmental stresses, such as heavy metals, when halophyte. It is salt-resistant and a hyperaccumulator compared to salt-sensitive crop plants. Thus, halophytes phytoremediator that can reduce organic and some may be the best candidates for phytoextraction or heavy-metal pollutants from industrial environments phytostabilization of heavy-metal-polluted soils and (Mohammadi Jahromi et al., 2019). Plants belonging to regions affected by salinity (Manousaki and Kalogerakis, the genus Salsola are dense summer annual halophytes 2011). The hypothesis is that tolerance to salt and heavy with rigid branches (Forbes and Allred, 1999), and they metals might be, at least partially, due to common are common in the arid and semiarid regions of our planet physiological mechanisms (Przymusinski et al., 2004). (Swart et al., 2003). Drought resistance mechanisms might indirectly Farzi et al. (2017) showed that three halophytic species, contribute to heavy metal tolerance, as heavy metal stress Salsola crassa, Salicornia europaea L., and Bienertia causes secondary water stress in plants in a manner similar cycloptera Bunge were salt phytoremediators. They grow to salt stress (Poschenrieder et al., 1989). Macnair (2003) well at all salinity levels and reduce salt to acceptable levels. suggested that metal accumulation in plants is related to Salicornia europaea, is a good candidate for effectively the biological evolution against drought. Water flow into * Correspondence: [email protected] 785

This work is licensed under a Creative Commons Attribution 4.0 International License. MOHAMMADI JAHROMI et al. / Turk J Bot the plant is responsible for translocation of heavy metals contamination in the soil. Disintegration and reduced into the tissues (Nedjimi and Daoud, 2009). size of the parenchymatous tissue, degraded and smaller Halophytes can be used to preserve the environment mesophyll tissue, reduced size of the xylem vessels, reduced or other organisms. For instance, they are helpful in root and stem diameter, and reduced leaf growth are some phytodesalination aimed at improving crop production, of the anatomical changes that occur. Moreover, there is a agricultural soil restoration, and sewage desalination spatial accumulation of heavy metals in different organs, (Suaire et al., 2016). According to Ravindran et al. commonly in the dermal, parenchyma, and phloem tissues (2007), Suaeda maritima L. (belonging to the family (Batool et al., 2015). As reported by Hameed et al. (2010), Amaranthaceae) removes salt from the soil and can salinity increases sclerification and succulence in the stem accumulate high quantities of salt inside its tissues. Salsola and leaves. soda L. successfully removes salt from the soil as well Due to lack of sufficient information on anatomical and (Karakas et al., 2017). structural aspects of S. crassa, and in light of its importance Salinity induces changes in plant organ morphology, for desalination and depollution, we set out to determine anatomy, and ultrastructure and has some physiological the anatomical structure of S. crassa and to describe how implications (Hameed et al., 2010). The appearance and much heavy metal toxicity causes specific structural and cell/tissue structure of root, shoot, leaf, fruit, seed, and functional modifications in this halophyte upon exposure flower may be affected by stress, or they become tolerant to industrial heavy-metal pollution. by adapting via a specific structure. Salt-accumulating glands, common in the Chenopodiaceae, remove excess 2. Materials and methods deleterious toxic ions and salt from photosynthetically active tissues and regulate ion concentration in different 2.1. Wastewater treatment methods plant tissues (Lefevre et al., 2009). These glands are not Young seedlings of Salsola crassa from Caryophyllales, always specific to Na and Cl; other toxic elements such the family Amaranthaceae taken from the fields of as Cd, Zn, Pb, Li, and Cu can accumulate in them as well Shiraz Industrial Park Refinery (Soltan Abad) in Shiraz, (Hagemeyer and Waisel, 1988). The glandular trichomes of a southern province of Iran (651558.86/3261713.68) were the leaf surfaces in Salsola paulsenni Litv. play a defensive identified for consideration as control and treatment plants. role against pathogens and environment, salt secretion, We selected 6 separately marked regions as treatment and pollination by producing different chemical products and control. The treatments were in 3, 2 × 2 m regions, (Mosyakin, 2017). Furthermore, other morphological and each was irrigated 3 times per week with 6–8 L of categories of Salsola species, such as nonglandular raw sewage around 12:00 PM. The controls were watered trichomes, scales or peltate hairs, and water vesicles which with the same volume of tap water at 12:00 PM. The soil are linked to secretion of salt and heavy metal removal characteristics, which were the same for the control and have been reported (Toderich et al., 2010). The leaves in treatment groups, and the heavy metal content of the Tamarix smyrnensis B. are covered by salt glands which water, soil, and wastewater are shown in Tables 1 and 2. accumulate and excrete Cd and Pb on the surface of the 2.2. Sampling and preparation for anatomical studies leaves, which indicates a salt excretion mechanism as After 3–4 months of irrigation the shoots, roots, and a possible detoxification mechanism (Kadukova et al., generative portions of the plants were dug out and 2008). Moreover, in Atriplex halimus L. leaves are covered subjected to the preparation steps for paraffin embedding by trichomes that collect high concentrations of Na+ and Cl−; when they rupture they leave salt crystals on the leaf (Ruzin, 1999). The process included fixation in FAA surface. Exposing these plants to Cd stress showed that (ethanol:formaldehyde:acetic acid; 17:2:1), dehydration in more than 30% of absorbed heavy metals were excreted ethanol and toluene, infiltration in toluene and paraffin, via the trichomes on the outside of the leaves (Lefevre and embedding in paraffin. Serial sectioning (5–20 µm, et al., 2009). The species of genusSalsola have extremely depending on the type of tissue) was carried out by rotary variable ecomorphological modes of photosynthesis and microtome (model MICROM HM 325, Thermo Fisher reproduction and grow well on saline or hypersaline soils Scientific, USA). Then, in order to perform histochemical (Akhani et al., 2007). studies, we used hematoxylin and eosin (H&E) and Heavy metal toxicity affects the growth and methylene blue-carmine alum staining (Ruzin, 1999) and development of agricultural crops by significantly altering mounted with Entellan. Observation and photography the physicochemical properties of soil: mainly organic were carried out using a Nikon light research microscope matter, cation exchange capacity, and pH. Degradation (E200, Nikon, Tokyo, Japan) attached to a digital camera of chloroplast and photosynthetic pigments and some (Samsung SCB 2000) with Stereolite software (Stereolite, anatomical changes may be induced via heavy metal Shiraz University of Medical Sciences, Shiraz, Iran).

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Table 1. Soil characteristics of the field.

CEC Sand Silt Clay O.C. O.M. T.N. EC Texture pH of paste (meq/100 g soil) (%) (%) (%) (%) (%) (%) (ms/cm) Soil sample 0.72 Loam 39.10 42.70 18.20 0.41 0.71 0.04 7.87 25.69

Table 2. Analysis of heavy metal pollution in the water, wastewater, and soil.

Fe (mg/L) Co (mg/L) Ni (mg/L) Zn (mg/L) Cu (mg/L) Pb (mg/L) Water 0.0260 0.0920 0.0890 0.0160 0.0080 0.0310 Wastewater 0.3990 0.1930 0.0530 0.0340 0.0180 0.0350 Soil* 17420.00 28.3500 90.6500 29.900 15.2500 2.200

*Amounts in the soil analysis are mg/kg.

2.3. Stereological studies white flowers in both samples, but more in the treatment We used random, systematic sampling to provide unbiased group. In addition, the Salsola flower is incomplete, since and quantitative data. The optical dissector setting included the petals disappeared into the main floral whorls, and an Eclipse microscope (E200, Nikon, Tokyo, Japan) only five petalloid sepals remained (Figure 3A). Sepals with a video camera that was connected to a monitor. A persist in the fruit, typically with wing-like appendages computer-generated point grid was superimposed on the (Figure 3B) that protect the inner structures of the bud screen using Stereology software system (Stereolite, Shiraz by covering them. Each of the five stamens is composed University of Medical Sciences, Shiraz, Iran). Quantitative of a filament and long anthers with four pollen sacs and information including thickness, diameter, length, numerous pollen grains inside. The single pistil in the width, and area of cells were calculated by the Stereology middle of the flower contains one ovule which develops program. For each characteristic, we first counted at least into a seed. It ends in two stigmas. The utricle, a large and 20 repetitions in the different sections, and then computed. dry fruit, is surrounded by five enlarged sepals, and appears Finally, we quantitatively compared the amount of each fan-shaped as it has well-developed wings (Figure 3B). The trait within each treatment. embryo is large and curved, with two cotyledons deeply divided or with an apical cleft, and without perisperm. In 3. Results late December the seeds can be harvested. 3.1. Morphological studies We irrigated the plants with waste and tap water Salsola crassa is an annual, strongly-branched plant (Figure in two separate groups. After applying the industrial 1). Over time, both the treatment and control Salsola wastewater for a month, morphology, such as color and plants underwent some changes in shoot appearance. The density, displayed the mildest impact. After three months stems, which were purple in color and quite succulent in of irrigation, the treated plants were a little paler and very young seedlings (March–April) (Figures 1A and 1B), thinner compared to the controls (Figures 4A and 4B). changed to very light green-to-silver, rigid and woody In the control plants, the fresh branches were denser and stems as time went on (Figures 1C and 1D). Occasionally, the leaves were greener with greater density and higher some changes in shoot color were observed during numbers; hence, internodes were shorter (Figure 4A). development (Figure 1E). The alternate dense young In comparison, the treated plants had spaced branches, leaves which were oval/ellipsoid, spherically ended, fleshy, while the stem was slightly pink, and the internodes were and thick became less dense and began to shrivel (Figures long with a few scattered leaves (Figure 4B). In terms of 1A–1F). In addition, their color changed from green to flowering time, the treated plants entered the reproductive greenish-silver over time (Figures 1A–1E). The young tap phase much later (about 1 month) than the controls. In root became completely wooden very quickly (Figures 2A general, flower and fruit sizes in the treated plants were and 2B). The tiny purple-pink flowers (Figure 3A) turned smaller (Figures 5A and 5B). into large, dark purple-red fruits in September (Figure 3B). 3.2. Structural studies The flowers were numerous, bisexual, and wind pollinated Microscopic sections showed some changes in plant as they are cross- or self-pollinated. We observed some structure after contamination, as follows.

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Figure 1. Salsola crassa development. From early vegetation (A) to late generative phase (F).

Figure 2. Root changes over time. (A) Thin tap root and (B) widely expanded woody root.

3.2.1. Stem and leaves among them; the latter increased over time. The appearance The outer layer of the stem consists of epidermis, with a few of the epidermis is very uneven with some crests (Figure caving stomata and trichomes, nonglandular and glandular 6A). The cortex can be divided into two regions. The outer

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Figure 3. (A) Small pink flower of S. crassa and (B) fan-shaped fruit.

Figure 4. (A) Control plant and (B) treated plant. region has a peculiar structure and is recognizable under part of the cortex among the parenchyma cells, there are the epidermis. It is a specific palisade chlorenchyma, which several small vascular bundles (Figure 6B) in addition to is discontinuous (Figure 6A) and consists of two parts: the a compact and conjoined vascular bundle seen at the end exterior part, which appears at first to have a cellular structure of the cortex (Figures 6C and 6D). The vascular cambium (palisade parenchyma enriched in chloroplasts) and an generates more xylem to the inside and less phloem to the inner part that contains small isodiametric cells. Below outside (Figure 6). Phloem, vascular cambium, and pith where this layer is formed, toward the cortex, degenerating with regular parenchyma cells are arranged in the stem layers of cells can be seen (Figure 6B). Then, parenchyma structure, respectively (Figures 6C–6F). cells appear in the inner cortex. The parenchyma is created In the treated plants, the outer layer of the stem by large cells with intercellular spaces. At the external epidermis was made up of cells non-uniform in size and

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Figure 5. (A) Salsola crassa fruit of control and (B) fruit of the treated plant. shape with more secretory glands and stomata (Figures differentiated into two regions; the outer region was 6B and 6G) than the control (Figure 6A). In the treated made up of a peculiar structure, which was the palisade plants, the specific layer of palisade chlorenchyma had chlorenchyma as seen in the stem, and the second, middle a narrower diameter than the control. The parenchyma region was composed of spongy parenchyma cells and cells in the cortex were irregular and deformed, and filled the inside of the leaves (Figures 7A–7D). The layer of sometimes the wall between the cells was lysing; however, palisade tissue had a cellular structure at first (Figure 7B), the controls were almost regular (Figures 6E and 6F). The but in the next stages of development, this changed into a treated parenchyma were heterogeneously larger, and their noncellular structure (Figures 7C and 7D). The primordia thickness was greater than in control. A number of small of the leaves were formed in bilateral arrangement (Figure vascular bundles that were separately spread among the 7E), and each held a large collateral bundle in the center parenchyma behind the epidermis of the treated sections between the two small vascular bundles (of the midrib were arranged as in the controls (Figures 6A and 6B); of primordium) consisting of xylem and phloem (Figure however, they were fewer in number. In the middle of 7F). Xylem was located at the upper side and phloem at the stem there was a pith with small intercellular spaces; the lower (Figure 7F). Fewer calcium oxalate crystals were however, the cells were smaller than in control (Figures 6E observed in the leaves in comparison with the stems. The and 6F). In some treatment cases, rupture was observed treatment sections also had these structures; however, they in the inner cortex tissue (Figure 6G). In the secondary also had a thinner palisade chlorenchyma layer and cuticle, structures of both control and treated samples, pith was as compared with the controls (Figures 7C and 7D). filled with bulky xylems and a large quantity of fibers 3.2.2. Generative organs (Figures 6H–6J). There were many calcium oxalate crystals The bisexual, incomplete flowers ofSalsola have both male in the vacuole of the pith cells of both samples (Figures 6K and female organs in the same flower (Figure 8A). The and 6L). anther of the stamen contained four pollen sacs, each with The alternate elliptical-shaped sessile leaves (Figure 1) two chambers, in all samples (Figure 8B). The outer layer of were externally covered by a thick cuticle. The epidermis all sections consists of epidermal cells (Figure 8C). Beneath tissue was made up of one layer of completely heterogeneous this layer, the endothecium cells form one layer. Inside this cells that gave it an uneven appearance (Figure 7A), caving layer, there are 2–3 parenchymatous middle layers (Figure paracytic stomata, salt glands, and ordinary epidermal 8C). The cells of the tapetum layer surround the pollen cells (Figures 7A–7C). The heterogeneous mesophyll sac. The pollen sac wall encloses a number of pollen grains

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Figure 6. Cross sections through S. crassa stem. (A) Control sample (×100). The partially formed subepidermal salt storage cells (SSC) and small vascular bundles (SVB) spread behind it. (B) Treatment sample (×100). Irregular appearance of epidermal cells (E) and parenchyma cells (P). (C) The vascular bundle of control sample consists of phloem (Ph) on the outside, vascular cambium (VC), and xylem (X) facing inside the cell. Note the thickness of the xylems (×100). (D) Vascular bundle of treatment sample (×100). (E) Section of control stem (×100). Note the almost regular parenchyma in the cortex. (F) Treatment stem (×100). Note the deformed parenchyma in the cortex. (G) Rupture between the cortex and the vascular cylinder in the stem (×100). (H) The pith and abundant fiber region (Fi) in the vascular cylinder can be observed (×100). (I) Note the small cortex volume versus the vascular cylinder in secondary structure of control sample (×100) and (J) treatment sample (×100). Note the xylem region with abundant fiber in the secondary structure.(K) Abundant calcium oxalate crystals in the pith of the control sample and (L) in the treatment sample (×100).

(Figure 8C). As time goes on, the tapetum layer disappears spherical, except for a few crescent ones; in the treated as it provides nourishment to the microspore mother plants, crescent, deformed, and unusually-shaped grains cells/microspores through secretion (Figures 8D–8F and were observed more often (Figures 9J and 9K). 9A). There are many pollen grains inside the pollen sac After fertilization, the ovary turns into a fruit. The fruit which develop into exine and intine with two nucleuses, is an utricle surrounded by five enlarged fin-shaped sepals vegetative and generative (Figure 9B). They are almost that form wings (Figure 3B). The outer surface of the wing spherical (Figures 9B and 9C) and numerous (Figure 9D). is colorful and looks like a beautiful dark-pink flower We observed some pollen in which tubes formed slightly (Figure 3B). The fruit contains one seed, which is rounded, (Figure 9E). During pollen grain development, the walls and a coiled embryo. The embryo has two cotyledons and between the chambers degenerate, and the chambers are is deprived of food reserves. joined together (Figures 9F and 9G). Then, a rupture 3.2.3. Stereological studies occurs, primarily at the end of the longitudinal wall of the The stereological studies confirmed our microscopic sac (Figure 9H); this rupture appeared earlier in treated observations. We calculated the average numerical value of samples. The pollen grains in the treated samples were parenchyma cell diameter of the control stem cortex (104 significantly fewer in number than in control (Figures 9D µm) and the treatment (81 µm) and the treatment stem and 9I). Likewise, their size was considerably smaller than cortex had decreased. The area of these cells decreased in the control group (Figures 9D and 9I). The tapetum as well (8366.4 µm control; 6431.4 µm treatment). The layer was thinner in the treatment group compared to the total size of each vessel bundle was also reduced (15.6 control (Figure 9). Pollen shape in the controls was almost µm control; 7.1 µm treatment). We saw a significant

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Figure 7. Salsola crassa leaf. (A) Appearance of the leaf (×40). (B) Palisade tissue on the outer side and spongy tissue in the center of mesophyll of a control leaf. Epidermis (Ep), palisade chlorenchyma (P.Ch), spongy parenchyma (S.P.), vein (V), and stomata (St) (×40). (C) Control leaf (×100). Primary palisade tissue replaced by the subepidermal salt-storage layer (SSL) at the latter developmental stage and (D) in the treated leaf (×100). (E) Leaf primordial (×40). The distal leaf is older in the leaf whorl (Num 1). The new leaf (Num 6) is near the apical meristem (AM). (F) A main vascular bundle (MVB) in the middle of the young leaf and two small vascular bundles (SVB) surrounding it (×40). decline in pith cell area, as well (2675 µm control; 990 efficiency, as Prasad and Strzalka (1999) mentioned, and µm treatment). The thickness of the peculiar layer of the represents the direct and indirect impacts of heavy metal palisade chlorenchyma in the treated stem sections was pollutants on photosynthesis. thinner (29 µm control; 25 µm treatment). In the leaves, 4.1. Stem and leaf structure the thickness of the cuticle (16.6 µm control; 7.02 µm The outer surface of the stem exhibits a number of crests. treatment) and the subepidermal layer (64.52 µm control; The outermost layer of cells in both stems and leaves— 29.70 µm treatment) both decreased significantly. epidermis—is covered by a thick cuticle, consisting of common epidermal cells, stomata, simple trichomes, and 4. Discussion salt glands that increases over time. The nonglandular Halophyte plants are resistant to salinization and drought trichomes on the outer surface of the epidermis are greater thanks to unique anatomical structures, which also make in number when the plant is younger, due to their protective them good candidates for resistance to other contaminants, role, and they are replaced by glandular trichomes over such as heavy metals, industrial and agricultural time. The salt glands were almost globular or club-shaped wastewater, and chemicals released from mines and the (Figures 7B–7D). The undulating epidermal surface oil–gas industries. Salsola crassa is a good example of with numerous salt-glandular structures had bulges with such a halophyte. It can survive in extreme environments deep grooves. These features were more significantly and face multiple stresses caused by high soil salinity, distinguishable in the leaves (Figures 7B and 7D). These long-term droughts, organic pollutants, and industrial results confirm the finding reported by Toderich et al. wastewater containing heavy metals. For these reasons, this (2010), which stated that there are vesiculated hairs in plant is considered an environmental phytoremediator, as some annual Salsola that are involved in cellular salt Mohammadi Jahromi et al. (2019) reported. Under such secretion. According to Luttge (1971), this may not polluted conditions, their morphology goes through be strictly a secretion process; salt glands may have a alterations, such as color changes, flower and fruit size specialized mechanism for removing salt from the plant. reduction, and reduction in branches and leaf density. Salt glands are usually spherical or club-shaped in Salsola This final change leads to a reduction in photosynthetic spp. and are distinguishable from unicellular papillae

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Figure 8. (A) Reproductive organs in S. crassa (×40). Ovary (OV), anther (An), leaflet (LfL).(B) Cross section of the male reproductive organ (×100). (C) Anther layers, respectively, epidermis (Ep), mechanical layer (ML), tapetum layer (TL). Pollen grain (PG) with exine is recognizable from outside to inside (×400). (D) Control pollen sac with some pollen grains (×400). The tapetum layer disappearing in late stages of development and (E) in the treatment (×400). (F) Pollen sac of treatment sample (×100). Deformed pollen grains can be seen. and sharp-pointed prickles (Toderich et al., 2010). The common, as it is involved in multiple roles including high- uneven epidermal surface with deformed and unequal capacity calcium regulation, heavy metal tolerance, and epidermal cells which we observed in treated stems is the protection against herbivorous (Nakata, 2012). Oxalate consequence of heavy metal pollution. Some researchers detoxifies Al3+ and regulates Ca2+ concentrations by found deformed and almost enlarged epidermal cells balancing soluble and insoluble forms as well as reducing resulting from encounters with heavy metals (Shah et al., the toxic effects of high Ca2+ concentrations. Moreover, 2010; Kouhi et al., 2016). research revealed that plant calcium oxalate crystals According to our results, the cortex consisted of caused stomatal closure that subsequently prevented water discontinuous peculiar cells of chlorenchyma (Figure loss under drought conditions (Tooulakou et al., 2016). 6A). These cells are composed of two types in the early In the vascular cylinder the larger xylem elements were developmental stage and are replaced by the integrated superficial, and the smaller ones were internal (Figures 6C tissue of salt-storage cells in the following developmental and 6D). The pith at the center of the stem is composed of phases (Figures 6A–6D). Bercu and Bavaru (2004) called large regular parenchyma cells (Figure 6H). This structure this the palisade cell layer. It is located compactly at the outer was reported in the same genus (Bercu & Bavaru, 2004). region of the cortex and contains numerous chloroplasts. Additionally, there were many calcium oxalate crystals in The continuity of this outer region is interrupted by large pith parenchyma (Figures 6K and 6L). collenchyma cells, and this structure exists in the leaves as The leaves were sessile and elliptical. The outermost well (Figure 7). We will discuss this issue in detail below. layer consisted of a uniseriate layer of common epidermal As shown in Figure 6B, the small vascular bundles were cells with paracytic stomata and glands. Furthermore, the unexpectedly recognizable below the peculiar layer and crested surface gave the epidermis an uneven appearance. among the parenchyma cells. The large parenchyma cells A thick cuticle covered the outer surface of the leaves in the cortex are involved in water storage. Calcium oxalate (Figures 7A–7C), and its thickness decreased in the treated crystals were observed in the parenchyma (Figure 6C). groups. The leaf structure of Salsola kali, reported by Bercu The existence of calcium oxalate crystal in plant tissues is and Bavaru (2004), was in line with our observations.

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Figure 9. (A) Pollen grain of treatment sample (×400). Note the secretory type. (B) The spherical exined pollen grains of the control sample (×400). Pollen grains (PG) attached to the tapetum layer (TL). (C) Round pollen grains of the control sample (×400). (D) Abundant pollen grains of the pollen sac of the control (×100). (E) Pollen grain of the control sample forming an early tube. Vegetative and generative nucleuses are present (×400). (F) Control pollen sac (×100) with degenerating wall. (G) Control pollen sac (×100) with numerous pollen grains. (H) Lysing end wall of the control pollen sac (×100). (I) Treated pollen grains (×100). (J) Crescent shape of treated pollen grains (×400). (K) Deformed pollen grains with exine can be observed in the treated sac (×100).

Based on our results, palisade tissue, which was third part of the stem as well, but discontinuously, as we gradually replaced by subepidermal salt-storage cells observed. The spongy parenchyma is located at the center located below the epidermis, formed the outer layer of of the mesophyll and is composed of large cells with a water the mesophyll (Figures 7B–7D). According to Toderich et storage role. This is in line with Grigore and Toma’s reports al. (2010), there are exclusive structures in Salsola species on Camphorosma annua P. (Grigore and Toma, 2007). such as swollen epidermal cells, a large-celled hypodermis, They also noted that the foliar limb ofSuaeda maritima and water-bearing parenchyma as well as subepidermal and the succulent tissues of Salicornia europaea L., which salt-storage cells that differ according to density of salt/ are from the same family and share anatomical similarities, ion deposits. Bercu and Bavaru (2004) considered this are related to the Sympegmoid anatomical type which chlorenchyma. In the early developmental stages, this contains superficial palisade cells with small and central layer is recognizable in two parts; the outer part consists fascicles included in the parenchyma. Bercu and Bavaru of longitudinal palisadic cells, and the inner part is made (2004) described the heterogenous mesophyll of Salsola up of small, isodiametric cubical cells. Grigore and Toma kali L. as differentiated into two regions: chlorenchyma (2007) reported two parts of a special structure in the foliar of palisade cells and parenchyma of spongy cells. They limb of Petrosimonia oppositifolia Litv., formed by the also reported that the palisade region is interrupted by epidermis. The outer, unilayered palisade tissue has very slightly sclerenchymatous cells. The treated chlorenchyma few cells and is called PCA tissue; its internal cells contain layer under the epidermis was thinner in comparison to a layer of assimilatory isodiametric cells called PCR tissue. the controls. Since this layer is involved in photosynthesis These layers are slightly discontinuous and define the and salt reserves, its thickness affects these phenomena. anatomical Kochioid type, which refers to the C4 pathway Heavy metals can have an impact on photosynthetic of the Kranz anatomy classification. Grigore and Toma functions, directly or indirectly (Prasad and Strzalka, (2007) reported that this peculiar structure occurs in the 1999), and the mechanisms of this impact include stomatal

794 MOHAMMADI JAHROMI et al. / Turk J Bot closure, change of chloroplastic structure, decreased less reproductive success in the next generation. Yousefi et photosynthetic pigments, decreased enzyme activities, and al. (2010) noted alterations in generative tissues affected by even modification of the spongy parenchyma structure heavy-metal pollution that are compatible with our results. (Gratão et al., 2009). According to our findings, there is only one pistil, As shown in Figures 7C and 7D, over time changes located in the middle of the flower, and its ovary contains occurred to the longitudinal palisade tissue, especially at just one ovule (Figure 8A). After fertilization, the ovule the tip of the leaf, which was replaced by a noncellular developed into a rounded seed. The utricle fruit was structure that appeared as salt particles, as reported by surrounded by five enlarged sepals, which gave it a Toderich et al. (2010). The existence of calcium oxalate in beautiful purple appearance (Figure 3B). the stem cortex and the leaves of some Chenopodiaceae The control and treated flowers were very similar in spp. (S. crassa, formerly classified into Chenopodiaceae) general; however, the treated flowers were smaller. Based was previously confirmed by a number of authors (Siener on Idzikowska, the pistil of S. kali comprises only one et al., 2006; Bercu & Bavaru, 2004; Dickie et al., 1989). embryo sac with a short style and a distal stigma. The To sum up, these structural modifications refer to stigma is a dry landing platform for pollen grains in the both plant physiological mechanisms—such as type of early stages and shrinks and dies after fertilization. While photosynthesis and harsh environmental conditions such the pistil forms the fruit, the anther and stigma dry and as high levels of salinity—and the impacts of pollution drop off the flower (Idzikowska, 2005). The fruit S.of kali on plant cellular structures, such as the appearance of is a vertical utricle surrounded by five enlarged fin-shaped deformed cells in all tissues. sepals. The internal surface of the wing is similar to the 4.2. Generative organs outer surface of the seed. However, the upper surface As described in Section 3, the Salsola crassa shrubs have forms the mature fruit as a beautiful flower (Idzikowska, small bisexual purple-pink flowers (Figure 3A). As the 2005). These statements are in line with our observation of plant branched out, the flowers were numerous during the S. crassa female reproductive organ. the blooming season (June–July). Four pollen sacs were located inside the five stamens of each flower. Each pollen 5. Conclusion sac was filled with abundant pollen grain (Figures 9B). We concluded that Salsola crassa has the morphology The descriptions of Salsola kali by Idzikowska (2005) and and anatomy of a halophyte. Succulent shoot appearance Chenopodium quinoa Willd. by Abdelbar (2018) were in includes huge water storage parenchyma cells in both line with our reports. the stem and leaves. Other structures such as peculiar As shown in Figure 8, the anther consists of a layer of palisade chlorenchyma changed into a salt storage layer epidermal cells, an endothecium of one layer, 2–3 layers over time beneath the epidermal layer of the stem and of parenchymatous middle layers, and, finally, the cells of leaves. Increased cuticle thickness and a sunken stomata, a tapetum layer surrounding the pollen sac. The pollen in addition to globular salt glands in the epidermis of sac wall enclosed a number of pollen grains (Figure 8). By the shoot structure—especially in the leaves—are other this time the tapetum layer had disappeared as it provided specialties of this succulent type which is resistant nourishment to microspore mother cells/microspores by to harsh environmental conditions. Salsola crassa is secretion (Figures 8D, 8E, and 9A). Many rounded and extremely resistant to wastewater treatment sewage spherical pollen grains developed in the exine and intine, enriched with heavy metals. These pollutants result in with two nucleuses, a vegetative and a generative (Figures changes in the morphology and structure of the plants, 9B and 9C). As Idzikowska stated, the male generative these changes are significant but not lethal. For example, organs in S. kali aggregates have five stamens differentiated low leaf density and scattered stem branches, fewer into anther, filament, and connective tissue. The mature flowers of smaller size, and smaller seed size are related anther is composed of four pollen sacs linked by connective to reduced photosynthetic efficiency which decreases tissue to the filament. Hence, the mature wall of the pollen the likelihood of reproductive success. Some structural sac comprises two layers, epidermis and endothecium, changes appeared in the treated group. These changes and the center is filled with numerous pollen grains. included reduced size of water storage parenchyma When the grains ripen, a rupture at the stomium leads cells in the cortex, pith, and vascular bundles; reduced to their release (Idzikowska, 2005); in the treated group, thickness of the cuticle in leaves; decreased thickness this occurred sooner. These reports are in line with our in the mechanical layers of the anthers; and diminished results. The earlier opening of the pollen sac wall, along number and size of pollen grains. These modifications with smaller and reduced numbers of pollen grains, and correlated to lower efficiency in tolerating drought the thinner tapetum layer in the treated samples leads to and salinity, as well as lower fertility. Furthermore, the immature or unsuccessful pollen grains, and this results in thickness of peculiar chlorenchyma under the epidermis

795 MOHAMMADI JAHROMI et al. / Turk J Bot in the stem and leaves decreased. Finally, the morphology Acknowledgments and structure of S. crassa plants may be affected by heavy The authors wish to thank Mr. H. Argasi at the Research metal contaminants in industrial wastewater; however, Consultation Center (RCC) of Shiraz University of the plant is resilient, the changes are not lethal; and, Medical Sciences for his invaluable assistance in editing therefore, it remains alive. this manuscript.

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