Anatomical adaptations in species of Poaceae growing in AlHa ' ir region of Riyadh , Saudi Arabia

The aim of this work was to determine the anatomical adaptations of leaves and stems of four species of Poaceae (Cynodon dactylon L. Pers., Chloris barbata SW. (Chloridoideae), Setaria verticillata L. P. Beauv., and Panicum coloratum L. (Panicoideae)) growing naturally at Al-Ha'ir region near Riyadh, Saudi Arabia. Cross-sections of the leaves revealed that the epidermis cells were spherical to oval and formed one layer with a thick cuticle as well as numerous bulliform cells in a fan shape and prickles. Ground tissue (mesophyll) consists mostly of chlorenchyma cells. A large vascular bundle surrounded by two bundle sheaths, outer sheath parenchyma and inner sheath sclerenchyma were observed in C. barbata and C. dactylon, while one bundle sheath of sclerenchyma surrounds the vascular bundle in S. verticillata and P. coloratum, while one bundle sheath with chlorenchymatous cells surrounded the small vascular bundles. Our results showed that all species contained a Kranz anatomy indicative of a C4 photosynthetic pathway despite belonging to two different subfamilies, Panicoideae and Chloridoideae. Oil droplets appeared in the mesophyll tissue of P. coloratum and S. verticillata. Crosssections of stems revealed an epidermis which consists of one layer of cells with spherical to oval shape and had a thick cuticle. Ground tissue contains strands of chlorenchyma cells followed by sclerenchyma tissue surrounding vascular bundles, thereby making a continuous cylinder. The vascular bundles were scattered in the ground tissue, with each vascular bundle surrounded by a single sclerenchymatous bundle sheath. Our results indicate that these plants were characterized by anatomical adaptations that enhance drought-tolerance capabilities, facilitating survival in arid and semi-arid regions such as Al-Ha'ir and thus these plants can be used to increase vegetation cover and pasture area in dry environments.


INTRODUCTION
The Poaceae family comprises a large number of economically important crops such as Triticum and Hordeum (Al-War'a et al., 1997), and several salinetolerant species, including Cynodon dactylon (Poljakoff-*Corresponding author. E-mail: aalshenaifi@ksu.edu.sa. Creative Commons Attribution e Author(s) agree that this article remain permanently open access under the terms of th License 4.0 International License Mayber, 1988) In addition, several plants in the Poaceae have important medicinal and aromatic benefits; for example, dried C. dactylon leaves are often used in anabolic and antiseptic substances (Gangwar et al., 2011), and the cereals S. italica (L.) P. Beauv is used to treat chicken pox in some areas of Pakistan (Ahmad et al., 2011). Moreover, C. barbata has been shown to have antibacterial activity (Natrajan et al., 2012). Several terrestrial plant species of family Poaceae were studied from the anatomical point of view (Kellogg, 2015;Panizzo et al., 2017). The plant species in our study were C. dactylon L. Pers. and Chloris barbata SW that belong to the Chloridoideae subfamily, while P. coloratum L and S. verticillata P. Beauv were part of the Panicoideae subfamily of the Poaceae family. These plants were considered permanent herbs, with the exception of S. verticillata, grow wild in Al-Ha'ir, an area south of Riyadh city in Saudi Arabia. However, the morphological characteristics of these species can vary significantly. Peng et al. (2017) studied the anatomical feature of swamp plant species that reflects the adaptation of plants to different environmental variables paralleled with morphological, eco-physiological and growth responses. Many studies have focused on the anatomical aspects of the Poaceae, such as Shaheen et al. (2012), who observed differences in leaf epidermal characteristics among six Panicum spp. native to Pakistan; these included variations in the shape of the phytoliths, prickles, large and small trichomes, and stomata, factors that were often used as important taxonomic indicators at the genus level. Ahmad et al. (2011) in a study of leaf epidermal characteristics of five plant species in Pakistan, including C. barbata and C. dactylon, found that differences in stomatal apparatus, subsidiary cells shape, the long epidermal cells, and silica cells were useful for distinguishing among species. Hameed et al. (2010) examined the anatomical adaptations of C. dactylon collected from saline and non-saline environments in Pakistan, reported that adaptations in plants growing in saline environments included saline excretions on the plant surface, accumulation of ions in the parenchyma tissue, reductions in stomata size on the leaf upper epidermis, increased bundle sheathing, and increased leaf bulliform cells size. Leaves epidermal characteristics of 13 species within 10 genera of Poaceae were studied by Nazir et al. (2013). They characterized the plants that grow in coastal zones by the presence of rows of silica bodies, which differed in shape and size among the plant species, silica bodies were cross-or dumb-bell-shaped or intermediate between the two and accompanied by cork cells. Vanesa and Cambi (2010) distinguished the occurrence of parenchyma tissue in the midrib region and prickles in leaves of Trichloris crinita, and further noted a set of subepidermal fibers alternating with chlorenchyma tissue, whereas in Pappophorum phillippianum and T. crinita, sclerenchyma cells were ring-shaped around vascular bundles. Eltahir and Abuereish (2010) reported the occurrence of small oil droplets in epidermal cells of leaf and mesophyll of Cymbopogon schoenanthus and Cymbopogon citratus . Carvalho Santos et al. (2013) evaluated variations in anatomical characteristics of 38 different genotypes of Brachiaria ruziziensis belong to Poaceae to identify genotypes that could be useful in plant breeding. Photosynthesis in C 4 plants was more active under environments that stimulate carbon loss through photorespiration, for example high light intensity and temperatures and reduced water obtainability in the case of stomatal closing (Carmo-Silva et al., 2009). The results of previous studies have shown that plants and grasses of the Poaceae were globally ubiquitous and often have great economic, medical and pastoral importance. Research has revealed numerous anatomical modifications among plants, which has greatly improved our understanding of how plants were able to overcome unsuitable environmental conditions and increase their growth and productivity. Our objective here was to determine the anatomical modifications of several of the plant species growing in Al-Ha'ir that contribute to their tolerance to the arid conditions of this part of Saudi Arabia. Improving our understanding of these adaptations may assist in enhancing vegetation cover in desert areas.

MATERIALS AND METHODS
The four plant species we focused on were members of the Poaceae, which grow naturally in Saudi Arabiathese species, consists of C. dactylon (L). Pers., C. barbata SW. (Chloridoideae), S. verticillata (L). P. Beauv. and P. coloratum L. (Panicoideae). Plant samples were collected at the flowering stage in the Al-Ha'ir region near Riyadh in the spring of 2017 and identified by botanists in the Herbarium, Department of Botany and Microbiology, King Saud University (KSUH). All samples were kept in 70% ethyl alcohol. Permanent sections were prepared in paraffin wax following the procedures described by Al-Khazraji and Aziz (1989) and Doaigey et al. (1997) for studying the internal anatomical characteristics of leaves and stems of the samples. Sections were examined and photographed with a TK-C1381EG video camera Japan, attached to an Olympus BX4LTF light microscope, Japan.

Leaf anatomical characteristics
Cross-section of the blades of C. dactylon, C. berbata, S. verticillate and P. coloratum leaves showed that the leaves were V-shaped, and the vascular bundles appeared in a single row parallel to the lower and upper surfaces of the leaf.

Upper epidermis
In leaves of all studied species, the upper epidermis consists of one layer of cells spherical to oval shaped, with thick outer walls, thin internal walls and protected with a thick cuticle. The epidermis cells in cross sections of C. dactylon appeared to be papillary shaped ( Figure  1b), and leaves of C. barbata contain many prickles (Figures 3a and 3b). In S. verticillata leaves, a thick cuticle covered the epidermis that contains bulliform cells (Figure 5a), whereas epidermis cells in leaves of P. coloratum were spherical to palisade-shaped, contained numerous large bulliform cells, and covered by a thick cuticle (Figures 7b and 7c).

Ground tissue
In C. dactylon ground tissue consists of an aerenchyma followed by 2 to 3 rows of chlorenchyma cells, filled with Banan et al. 203 chloroplasts and surrounded by thin cellulose walls ( Figure 1b). The ground tissue in C. barbata, consists of 4 to 7 irregular-and large-sized parenchyma cells with thin cellulose walls free of chloroplasts, followed by 2 to 3 layers of chlorenchyma cells Containing chloroplasts ( Figure 3a). Ground tissue in S. verticillata consists of 2 to 10 layers of irregularly-shaped parenchyma cells with thin cellulose walls free of chloroplasts followed by 2 to 3 layers of chlorenchyma cells filled with chloroplasts ( Figure 5a). Finally, in P. coloratum, the ground tissue consists of 4 to 6 layers of irregularly shaped parenchyma cells, free of chloroplasts, with thin cellulose walls followed by 2 to 3 layers of chlorenchyma cells filled with chloroplasts (Figures 7a and 7b).

Vascular tissue
Vascular tissue in the leaves of these species consists of oval-shaped, closed collateral bundles in oval shape. The bundle consists of xylem that is oriented towards the upper epidermis in an approximately V-shape and contains large cavities and thick lignin walls in vessels, with an inverted metaxylem occupying the V-shape head. The phloem, containing companion cells and sieve tubes, was located near the lower epidermis. Large vascular bundles were surrounded by two layers of cells that define the perimeters of the bundle sheaths; its outer layer contained large parenchymal cells, containing some chloroplasts, and surrounded by cellulose thin walls. However, the inner layer was composed of small lignified thick-walled sclerenchyma cells. In the leaves of all the four plants a bundle sheath of parenchyma cells containing chloroplasts were surrounded the small vascular bundles (Figures 1b, 3b, 5a and 7b). Large vascular bundles were connected to the lower epidermis via a group of sclerenchyma cells with lignified walls; sclerenchyma cells in 3 to 5 layers were presented in C. dactylon and P. coloratum (Figure 7a), 2 to 4 layers occur in S. verticillata ( Figure 3b) and 5 to 7 layers were found in C. barbata (Figure 5a).

Lower epidermis
It was similar in appearance to the upper epidermis in leaves of all four plants, except that prickles were absent in the leaf epidermis in C. barbata (Figure 3b).

Ground tissue
Ground tissues in all plants had 3 to 4 layers of chlorenchyma cells, filled with chloroplasts, and surrounded by thin cellulose walls. The results showed that C. dactylon (Figure 1c) was different compared to C. barbata (Figure 3c) in the presence of aerenchyma, whereas S. verticillata ( Figure 5d) and P. coloratum ( Figure 7d) were dissimilar in the occurrence of oil droplets in some cells.

Lower epidermis
It was similar to the upper epidermis of the four plants under study in its Characteristics, except the absence of bulliform cells and a thick cuticle in C. dactylon ( Figure  1c), C. barbata (Figure 3c), and S. verticillata (Figure 5c). The lower epidermis in ground tissue of P. coloratum consisted of a single layer of spherical-to palisadeshaped cells, with thick outer walls covered by a thin cuticle and thin internal walls (Figure 7c).

Epidermis
Stem epidermis was composed of a single layer of spherically-to elliptically-shaped cells with thick outer and thin internal walls. Outer walls were covered by a thick cuticle in C. dactylon ( Figure 2a) and C. barbata ( Figures  4a and b), but the cuticle was thin in S. verticillata ( Figure  6a) and P. coloratum (Figure 8a).

Ground tissue
For C. dactylon, the results showed that ground tissue (chlorenchyma tissue) consists of 2 to 3 layers of thinwalled parenchyma cells containing chloroplasts followed by 3 to 5 layers of sclerenchyma cells in a cylinder shape, which was connected to the external vascular bundle sheath. The remaining ground tissue consists of parenchyma cells free of chloroplasts and with thin cellulose walls, with cell size increasing towards the center of the pith and containing large intercellular spaces (Figures 2a and 2b). Ground tissue in C. barbata was composed of 3 to 4 layers of chlorenchyma cells with thin cellulose walls, and containing chloroplasts. Clusters of 3 to 4 layers of sclerenchyma cells with lignin walls were connected to the epidermis, and were followed by 3 to 4 layers of sclerenchyma cells that form a cylinder shape. The sclerenchyma cylinder connects to the sheath of the vascular bundle. The remaining ground tissue consists of thin-walled parenchyma cells free of chloroplasts, with cell size increasing towards the center of the pith, and with clear intercellular spaces (Figures 4a  and 4b). In S. verticillata, ground tissue consisted of 4 to 5 layers of small parenchyma cells with thin cellulosic walls, which represent the initial ground-tissue layers, along with 3 to 4 layers of sclerenchyma cells with lignified walls, which were connected to the vascular bundles. The ground tissue towards the pith center (Figure 6a and 6b) was similar to that in C. barbata; however, crystalline sand was also found in the parenchyma cells (Figure 6c). Finally, in P. coloratum, the ground tissue consisted of 4 to 5 layers of chlorenchyma cells with thin cellulose walls containing chloroplasts, along with 4 to 5 layers of sclerenchyma cells that form a complete cylinder, which connect to the sheaths around the vascular bundles. The remaining ground tissue in this species consists of parenchyma cells free of chloroplasts with thin cellulose walls, with cell size increasing towards the center of the pith and containing small intercellular spaces (Figure 8b).

Vascular tissue
Vascular bundles were scattered throughout the ground tissue in all four species, and the stem center was free of vascular bundles, except in P. coloratum. The vascular bundles form closed collateral and were oval shaped. Vascular bundles consist of xylem and phloem, with the primary xylem including metaxylem vessels, the latter was located at the head of the V-shape, and vessels with thick lignin walls and large cavities, and protoxylem vessels were replaced by air spaces or protoxylem cavities. The phloem was located toward the outer side and contained companion cells and sieve tubes. The vascular bundles were distributed throughout the primary tissue in all four plant species and were surrounded by sclerenchyma cells in one layer (that is, a bundle sheath;

DISCUSSION
Our examination revealed several of the internal anatomical adaptations of the leaves and stems associated with higher drought tolerance in C. dactylon (L). Pers., C. barbata SW, S. verticillata (L). P. Beauv. and P. coloratum L (Figures 1 to 8). As shown in Figures  1, 3, 5, and 7, the leaf blade was narrow and V-shaped in C. dactylon and C. barbata (Figure 1a and Figure 3a), but V-shaped and divergent in S. verticillata and, P. coloratum (Figure 5a and Figure 7a). Our results accord with those of several previous studies in which it was suggested that the U-shaped (Metcalfe, 1960) or Vshaped (Doğan and Tosunoğlu, 1992;Mavİ et al., 2011) leaf blades of members of the Poaceae were an adaption to reduce the leaf-surface exposure to light, thus reducing water loss; moreover, the flat blade may facilitate absorption of vital minerals at times when water was plentiful.
Our analyses of leaf cross-sections (Figures 1, 3, 5, and 7) indicate that the epidermis was covered with a thick cutin cuticle, which aids in water retention. The presence of stomata on both epidermal leaf surfaces, as well as the large intercellular spaces in leaf mesophyll tissue, facilitates gas exchange. Thin-walled bulliform cells with large vacuoles were plentiful in the upper epidermis above the prominent midrib region, and play a role in the folding or shrinking of the leaves when plants were under water stress, which reduces the amount of exposed leaf surface area and lowers rates of transpiration and light absorption. These results were consistent with the reports of Carmo-Silva et al. (2009), Gangwar et al. (2011) and Grigore and Toma (2017). In addition, the presence of different forms of silica cells, cork cells with suberized walls, and sclerenchyma cells in bundles that link with the epidermis via the large vascular bundles, were all traits that increase the strength and    bundles in an oval shape, with each bundle surrounded by two bundle sheaths composed of radially-shaped cells (chlorenchyma outside and sclerenchyma inside), whereas in S. verticillata (Figures 5a and b) and P. coloratum (Figures 7a and b), each vascular bundle was surrounded by a single bundle sheath composed of sclerenchyma cells and partially surrounded by chlorenchyma cells. In all four species, lateral vascular bundles were surrounded by a single bundle sheath of parenchyma cells, even though the four species belong to different genera and sub-families (Chloridoideae and Panicoideae).
Leaf blades contain a large number of vascular bundles, reflecting a greater ability to absorb water (Carmo-Silva et al., 2009), moreover, we observed oil droplets in the ground tissues of P. coloratum (Figure 5d) and S. verticillata (Figure 7d) leaves. Such oil droplets, which reduce water loss and can serve as an energy source  (Eltahir and Abuereish, 2010), have been found in other epidermal cells of Poaceae species. Analysis of the stem cross-sections indicated (Figures 2, 4, 6, and 8) the presence of thick epidermal cellular walls due to an abundance of sclerenchyma tissue, which assists in the regulation of water loss, along with the presence of parenchyma cells that make up the ground tissue, which increases water storage capacities. We observed an abundance of vascular bundles in the ground tissues and metaxylem elements, which serve to enhance transport efficiency and provide maximal amounts of water and minerals to the vegetative parts of the plant for the proper functioning of photosynthesis and other biological processes. Moreover, the presence of many sieve tube elements in the phloem also improves nutrient transport (Hameed et al., 2010) and storage.
The results of our analysis indicate that the internal structures of the leaves and stems of these four plants exhibit a variety of adaptations that enhance drought tolerate capacity. These anatomical adaptations allow these species to thrive in the arid and semi-arid regions of Saudi Arabia, and can be used to increase vegetation cover and increase pasture area.