Journal of Novel Applied Sciences Available online at www.jnasci.org ©2016 JNAS Journal-2016-5-6/204-212 ISSN 2322-5149 ©2016 JNAS TYPES OF MESOPHYLL IN ASSIMILATING ORGANS IN CONNECTION C3 AND C4 PHOTOSYNTHESIS TYPES IN THE SPECIES OF GENUS CLIMACOPTERA BOTSCH. (CHENOPODIACEAE VENT.) CENTRAL ASIA Guljan M. Duschanova Academy of Sciences, Republic of Uzbekistan, Institute of Gene Pool of Plants and Animals. 232 Bagishamol Str., Tashkent, 100053 Corresponding author: Guljan M. Duschanova ABSTRACT: The structure of assimilating organs in five species of Сlimасорtеrа is described: mesophylls of cotyledon, leaf, bract and perianth. The change type mesophylls assimilating organs from the cotyledons (and leaf bracts, bracteoles) to perianth in species Climacoptera on saline soil and Mirzachul (Uzbekistan, Province Sirdarya) and Kyzylkums (Uzbekistan, Province Buhora, Navoiy, Karakalpakstan, Province Miskin) related C3 and C4 types of photosynthesis. Identified quantitative parameters Kranz-cells and their variability in the ontogeny of plants and species specificity. The level of their specialization, xeromorphy and halomorphy is determined. Regularities of adaptation of succulent species with the Kranz anatomy to xero- and halo factors, which enable insights into the nature of their halophytism, are revealed. A high coefficient of the similarity of species from one section in different habitation conditions was shown using the cluster analysis. Keywords: adaptation, assimilating organs, Climacoptera, halophyte, xerophyte, Mirzachul, Kyzylkum, Kranz-anatomy. INTRODUCTION In some Chenopodiaceae species the combination of С3 and С4 types of photosynthesis is observed during ontogeny. This is connected with the structure of different assimilating organs. The structure of leaves in some species of the order and ecological groups of Chenopodiaceae has been studied for a long time due to the processes of their adaptation and in the last few years due to the type of metabolism (Carolin et al. 1975; Butnik et al., 1991; Fisher et al. 1997; P’yankov et al. 1997). Butnik et al. (1991) indicated the necessity of the study and comparison of the cotyledon structure with that of the leave, i.e. changes in the mesophyll type in ontogeny. Further studies by V. P’yankov et al. (1998), E. Voznesenskaya et al. (1999), H. Freitag, W. Stichler (2000) and Butnik (1999) indicated the promising feature of comparison of assimilating organs for the understanding of the evolutionary level of a taxon and changes in the type of metabolism. In this connection it is important to identify in which organs and at what stages of ontogeny Kranz cells are present. The goal of the work is to reveal types of mesophyll in vegetative organs, which perform the assimilating function at different levels of plant ontogeny. J Nov. Appl Sci., 5 (6): 204-212, 2016 MATERIALS AND METHODS Assimilating organs in five species of the genus Climacoptera species from three sections were examined: 1. Section Ulotricha Pratov. – Climacoptera ferganica (Drob.) Botsch. is an annual plant, euhalophyte, grows in solonchaks (saline soils), takyrs (dry-type playa) and saline sands. Geographical range: Iran, Afghanistan, Central Asia, and Dzungaria. 2. Section Amblyostegia Pratov. – Climacoptera intricata (Iljin) Botsch. is an annual plant, euhalophyte, grows in solonchaks (saline soils). Endemic to Central Asia. – Climacoptera aralensis (Iljin) Botsch. is an annual plant, euhalophyte, grows in solonchaks, saline sands. Endemic to Central Asia. 3. Section Climacoptera Pratov. – Climacoptera lanata (Pall.) Botsch. is annual plant, euhalophyte. The species with a wider area: Turkey, Iran, Afghanistan, Pakistan, northeastern area of Russia, Central Asia, Dzungaria and therefore more diverse ecology, solonchaks (saline soils), pit-run fines-gravelly soils, saline sands. – Climacoptera longistylosa (Iljin) Botsch. is an annual plant, grows on saline soils. Natural habitat: Afghanistan, Central Asia and Kazakhstan (Pratov, 1986). C. intricata and C. longistylosa grow in Mirzachul (Uzbekistan, Province Sirdarya) in compacted less saline soils, C. ferganica, C. lanata and C. aralensis grow in southwestern Kyzylkum (Uzbekistan, Province Buhora, Navoiy and Karakalpakstan, Province Miskin) on less compacted sandy loam, but more saline soils. The mesophylls of cotyledons and leaf (from the mid-layer of annual shoots), bract, bracteole and perianth in the beginning of flowering were studied. The material collected for the study was fixed in 70° ethyl alcohol. The type of mesophyll was identified on cross-sections. The sections were made using the straight razor, dyed in the methylene blue, glued in glycerol- gelatin. The form and shape of major epidermal cells were described using the methods described by Zakharevich (1954). Measurements were made using the routine methods (Barykina, Chubatova, 2005). The preparations were drawn by using the drawing apparatus RA-6 under the microscopy MBI3. The measurements were replicated 30 times and the average value was calculated; errors of the measurement and significance were identified using Zaitsev formula (1991). The cluster analysis was conducted using the software Past on the basis of Jaccard index of similarity (Shmidt, 1984). RESULTS AND DISCUSSION Cotyledons in the species of the genus Climacoptera are lamellate, oval. The longest and widest are recorded in C. intricata and C. longistylosa; the small ones in C. ferganica and C. lanata, C. aralensis. The mesophyll in cotyledons is dorsoventral, of the non-Kranz type, with one row of palisade cells (C. ferganica, C. longistylosa and C. aralensis), two rows (C. intricata) or 2-3 rows (C. lanata). The tallest palisade cells were recorded in C. Ferganica and C. aralensis; the lowest in C. lanata (Table 1, Fig. 1). The cells of the lacunose parenchyma were largest in C. intricata, while smallest in C. lanata and C. aralensis. Conducting bundles in C. ferganica and C. aralensis are not scleral; in other species are weakly scleral and surrounded by one row of parenchymal liner. Mesomorph traits prevail in the structure of cotyledons: the absence of indumentums and a low palisade index. Table 1. Quantitative values of the cotyledons of the Climacoptera species (n=30) Value Cotyledons: length, mm width, mm thickness, µm Epidermis: height, µm Thickness of outer wall, µm Palisade cells: number of rows layer thickness height, µm width, µm palisade index Lacunose parenchyma: number of rows thickness, µm d – cells, µm Number of conducting bundles C. ferganica C. lanata C. intricata C. ongistylosa C. aralensis 5,8±0,2 1,5±0,02 338,2±2,3 8,8±0,2 3,4±0,02 297,3±2,8 11,2±0,2 4,8±0,2 321,7±8,1 10,8±0,2 4,8±0,2 284,0±4,4 5,2±0,2 1,3±0,02 264,1±2,4 31,0±0,4 26,9±0,3 28,9±0,8 28,3±0,6 27,4±0,2 5,4±0,1 5,1±0,1 4,4±0,1 4,5±0,1 4,6±0,1 1 40,0±0,7 40,0±0,7 18,5±0,4 2,1 3 72,1±0,8 29,0±0,5 17,1±0,2 1,7 2 68,2±1,3 30,1±0,9 22,9±0,5 1,3 1 32,3±0,5 32,3±0,5 14,3±0,2 2,2 1 40,5±0,4 40,5±0,4 17,7±0,2 2,3 8-10 236,2±0,9 34,2±0,3 11,1±0,2 6-7 171,4±1,3 25,5±0,3 17,4±0,2 7-8 186,2±2,4 34,5±0,5 8-9 195,1±2,7 28,4±0,5 21,6±0,2 8-10 204,0±1,9 25,3±0,2 8,9±0,1 22,2±0,2 205 J Nov. Appl Sci., 5 (6): 204-212, 2016 Fig. 1. Structure of the cotyledons in the species Climacoptera on the cross-section: a – a – C. ferganica; b – b – C. lanata; c – c – C. 1 2 1 2 1 1 2 2 intricata; d – d – C. longistylosa; e – e – C. aralensis. a – e – scheme, a – e – detail. Key: P – palisade parenchyma, spongy parenchyma, CB – conducting bundle, E – epidermis. 1 2 1 2 1 2 The species of the genus Сlimасорtеrа are typical leaf succulents. Adaptive traits of the succulent group are the presence of a specialized water bearing tissue, performance of water- and salt-retaining function by other leaf tissues, including the epidermis. The analysis of quantitative indexes of the leaf revealed characteristic traits of species of the genus Сlimасорtеrа Воtsch. The largest leaves are in С. longistylosa and C. intricata, the small, С. ferganica, C. lanata and C. aralensis. The leaf is covered with three types of trichomes: long 3-8 cellular monostich us trichomes with a broad basis, falling; short 1-2 cellular with narrow basis (3 species); thin numerous long filiform of a single type (C. intricata). Least covered were the leaves of C. ferganica (Table 2). Two Kranz types of mesophyll were found in the leaf: Kranz-ventro-dorsal mesophyll at the basis; close to Kranz-centric (Salsoloid - type) but differing from it by the openness of chlorenchyma on the adaxial side and separation of conducting bundles from the Kranz lining by water-bearing cells. As G. Kadereit, T. Borsch, K. Weising et аl. (2003), we believe that this type of mesophyll as an independent Сlimасорtеrа-type (Fig. 2). 206 J Nov. Appl Sci., 5 (6): 204-212, 2016 Table 2. Quantitative measures of leaves of Climacoptera species (n=30) Indicator Leaf: length, cm width, mm thickness, mm Epidermis: height, µm thickness of outer wall, µm area of cell, µm2 Palisade parenchyma: height, µm width, µm index of palisade layer Waterbearing cells, µm: layer thickness % of d – leaf d-cell number of rows Kranz-lining, height, µm Number of vascular bundles Number of vessels in the main vascular bundle Diameter of vessels, µm Species C. ferganica C. lanata C.intricata C. longistylosa C. aralensis 19,8±0,6 1,8±0,04 1,28±0,01 21,8±0,8 2,2±0,07 1,46±0,02 30,0±0,8 1,9±0,04 1,43±0,01 31,6±0,5 2,4±0,7 1,33±0,01 17,4±0,3 1,6±0,04 1,1±0,01 37,0±0,3 32,1±03 45,1±0,4 31,4±0,3 40,0±0,5 4,3±0,1 2908±38,5 3,7±0,1 2155±19,1 7,5±0,1 3610±52,4 4,5±0,1 3621±39,7 5,8±0,1 2890,6±50,3 49,2±0,4 14,0±0,2 56,3±0,6 12,0±0,2 50,0±0,3 13,3±0,2 65,4±0,4 15,1±0,1 49,4±0,3 13,6±0,2 3,5±0,06 4,7±0,08 3,8±0,06 4,3±0,04 3,7±0,1 1197,4±4,1 95 189,8±4,3 6-7 18,3±0,2 25 14 9,0±0,2 1180,9±7,8 91 138,5±3,9 7-8 16,0±0,1 23 16 9,1±0,2 1316,2±8,2 89 200,3±5,3 6-7 28,0±0,3 23 18 15,8±0,3 1087,7±7,3 88 218,8±3,2 5-6 19,3±0,4 24 24 9,9±0,2 1245,2±7,3 91 332,2±2,7 4 19,1±0,1 21 23 11,3±0,3 Fig. 2. Structure of the leaves in the species Climacortera on the cross-section: a, a – C. ferganica; b – C. 1 lanata; c – C. intricata; d – C. longistylosa; e – C. aralensis. a – e – detail, a – schemes of different parts of leaves. Key: W – water-bearing parenchyma, D – druse, KL – Kranz-lining, P – palisade parenchyma, CB – conducting bundle, PCB – peripheral conductive bundle, E – epidermis. 1 207 J Nov. Appl Sci., 5 (6): 204-212, 2016 The combination of xeromorphic and halomorphic signs is observed in all types of leaves, which are manifested in different combinations. Xeromorphic signs in C. Ferganica are small vessels in the conducting bundle; a high palisade index, smaller water-bearing cells and Kranz-lining, small vessels in conducting bundle in C. lanata; in C. longistylosa, high palisade cells, high palisade index, dense indumentum from numerous thin trichomes, significant height of epidermal cells with a thickened exernal wall (C. intricata and C. aralensis). Halomorphic signs of leaf are present in all species in different combinations: a scarce indumentums, thick water-bearing layer; in C. intricata large water-bearing leaves, high Kranz lining and large vessels; in C. longistylosa and C. aralensis water-bearin g cells, numerous vessels in conducting bundles (Fig.4, Table 4). In general the leaves of C. intricata, C. longistylosa and C. aralensis are characterized with a high succulent feature, while ferganica and C. lanata, with a xeromorphic feature. We considered quantitative traits of the leaf in relation to the ecology of the habitat and sectional belonging. The quantitative indices of the leaf length, thickness of water-bearing layer, height of Kranz lining, vessel diameter in the main conducting bundle are close in the species from different sections, however growing in similar conditions. As a generative organ, the bract protects the flower from the environmental effects. The bract, bracteole and leaves of perianth in some species (C. intricata, C. lanata and C. longistylosa) were studied by D.T. Khamraev (2006) and their diagnostic traits were revealed. We for the first time studied the structure of the bract, bracteole and leaves of perianth only in C. aralensis. The bract in C. aralensis is very succulent, a little blunt-pointed, egg-like. Contrary to the leaf, the bract and bracteole are pubescent with short prominent hair. Epidermal cells are rounded and oval. The mesophyll is Kranz ventrodorsal in the bract and bracteole; in cross-section it has the shape of a crescent as in the leaf base. On the adaxial side, the water-bearing parenchyma borders on the epidermis; the palisade parenchyma and Kranz lining are situated on the abaxial side. The palisade parenchyma is smaller than in the leaf in the mid-part of the bract and bracteole. The Kranz lining consists of cubic cells. The number of rows of water-bearing cells in bract and bracteole is 3-4; in the leaf, 5-8. Three conducting bundles lie in the center of the leaf, while in bract and bracteole only one. The main vein is situated in the center of the cross section. In the leaf, the central conducting bundle contains more vessels than in the bract and bracteole (24 and 17-18, respectively). The peripheral conducting bundles are numerous (20); similarly to the leaf they are separated from the Kranz-lining by 3-4 rows of water-bearing cells, which contain with druses of calcium oxalate (Fig. 3; Table 3). The structure of the lower part of leaves of perianth in Climacoptera brachiata and C. lanata was described by A.A. Butnik (1972), C. ferganica, by S.A. Paizieva (1983). Their significant parenchyma growth was noted. The leaves of the perianth in C. aralensis are lancet-shaped and pubescent with short unicellular hair. The cells in the inner (adaxial) is smaller than the exterior (abaxial) epiderm of the perianth; their outer walls are thickened to 4,9±0,1µm. The height of the epidermis is 19,7±0,3 µm. The mid-part of the mesophyll leaves of the perianth consists of five rows of parenchyma, of which three are large and two are small. The thickness of the parenchyma layer in the cross section reaches 78,4±0,3 µm; the diameter, 34,1±0,3 µm. The number of conducting bundles varies from 6 to 10. The central conducting bundle is large; it contains 8-10 small vessels. In mid- and lower part of ЛОЦ some cells in all tissues are filled with druses of calcium oxalate. The bract and bractioles protecting the flower have a specialized structure similar to that of the leaf. The perianth is deprived of chlorenchyma. 208 J Nov. Appl Sci., 5 (6): 204-212, 2016 209 J Nov. Appl Sci., 5 (6): 204-212, 2016 Table 3. Quantitative indices of Climacoptera aralensis structure (n=30) Organ / tissues Bract Epidermis, µm: Indices height thickness of outer wall Palisade parenchyma: height, µm width, µm palisade index Water-bearing cells: thickness of water level, µm d-cells, µm number of cell rows Kranz-lining: height, µm The number of conducting bundle Number of vessels in the conducting bundle Vessel diameter, µm Bracteole Epidermis, µm: height thickness of outer wall Palisade parenchyma: height, µm width, µm palisade index Water-bearing cells: thickness of water level, µm d-cells, µm number of cell rows Kranz-lining height, µm The number of conducting bundle Number of vessels in the main conducting bundle Vessel diameter, µm Leaf of perianth Epidermis, µm: height thickness of outer wall Parenchyma: thickness, µm d – cells, µm number of cell rows The number of conducting bundle Number of vessels in the main conducting bundle 33,1±0,3 3,5±0,08 35,4±0,3 10,5±0,2 3,4±0,05 11239±3,7 138,5±3,9 3-4 12,4±0,1 20(1) 17-18 6,5±0,1 26,7±0,2 4,9±0,1 42,7±0,4 13,3±0,2 3,2±0,1 814,9±2,3 177,4±1,4 3 19,3±0,2 17(1) 16-17 6,0±0,1 19,7±0,3 4,9±0,1 78,4±0,3 34,1±0,3 5 6-10 (1) 8-12 The cluster analysis of the quantitative indices of the cotyledon of species from different sections C. ferganica (section Ulotricha) and C. lanata (section Climacoptera) from the Kyzylkum revealed a smaller coefficient of trait similarity (0,15) than in C. intricata (section Amblyostegia) and C. longistylosa (section Climacoptera) from Mirzachul (0,28). The species C. lanata и C. longistylosa belonging to one section, but growing in different conditions showed a lower coefficient of similarity (0,11) than the species C. intricata and C. longistylosa from different sections. This suggests a significant impact of habitats on the formation of shoots and the relation to the endemism of species in Mirzachul (Table 4, Fig. 4). Table 4. Jaccard coefficient (Кj) of species Climacoptera By the quantitative indices of cotyledons Indicators and number of species 1- C. ferganica 2 - C. lanata 3 - C. intricata 4 - C. longistylosa Кj C. ferganica 0,15 0,21 0,17 C. lanata 0,15 0,11 0,11 C. intricata 0,21 0,11 0,28 C. longistylosa 0,17 0,11 0,28 - 210 C. lanata 1 C. intricata 1 4 3 2 J Nov. Appl Sci., 5 (6): 204-212, 2016 C. longistylosa C. ferganica 0,9 b 0,8 Similarity 0,7 0,6 a 0,5 0,4 0,3 c 0,2 Fig. 4. Cluster analysis by Jaccard similarity coefficient (Кj) in Climacoptera species by quantitative measurements of cotyledons. Abbreviations: а = Section Amblyostegia Pratov.; b = Section Climacoptera Pratov.; с = Section Ulotricha Pratov. The cluster analysis established the coefficient of similarity of leaf traits was 0,17 in C. intricata - C. longistylosa from different sections in similar conditons (Mirzachul). This value in plants from different sections but in similar conditions (Kyzylkum) showed the following: in C. ferganica-C. lanata – 0,19, in C. ferganica - C. aralensis – 0,17. In C. lanata - C. longistylosa, from one section in different ecological conditions, this value reached 0,24; in C. intricata-C. aralensis from one section in different ecological conditions – 0,23 (Table 5, Fig. 5). Table 5. Jaccard coefficient of similarity (Кj) in Climacoptera species by quantitative measurements of leaves C. lanata C.longistylosa 0,14 0,24 0,17 0,10 C.aralensis 0,17 0,10 0,23 0,10 - C. longistylosa C. ferganica 1 C. aralensis C. intricata 0,12 0,14 0,17 0,23 4 3 C. intricata C.lanata 0,19 0.14 0,24 0,10 2 Кj C.ferganica 0,19 0,12 0,14 0,17 5 Indicators and number of species 1-C. ferganica 2-C. lanata 3-C. intricata 4-C. longistylosa 5-C. aralensis 1 0,9 0,8 0,7 Similarity 0,6 0,5 0,4 0,3 0,2 a 0,1 b c Fig. 5. Cluster analysis by Jaccard similarity coefficient (Кj) in Climacoptera species by quantitative measurements of leaves. Abbreviations: а = Section Amblyostegia Pratov.; b = Section Climacoptera Pratov.; с = Section Ulotricha Pratov. 211 J Nov. Appl Sci., 5 (6): 204-212, 2016 CONCLUSION Thus, the study of assimilating organs in five species of the genus Сlimасорtеrа revealed Kranz (the leaf, bract and bracteole) and non-Kranz (cotyledon and perianth) structure types, as well as the level of their xeromorphic and halomorphic traits connected with ecological conditions of habitation. A similarity of many indices of assimilating organs; the genotypic component is manifested more significantly. A considerable level of similarity of C. lanata – C. longistylosa (section Сlimасорtеrа) and C. intricata – C. aralensis (section Amblyostegia), in comparison with C. intricata and C. longistylosa is perhaps attributable to to their plasticity under conditions of a larger habitat. The species C. intricata, C. aralensis (endemic to Central Asia) and C. longistylosa (Central AsiaAfghanistan) were perhaps formed later under the conditions of arid climate in a narrower ecological range. 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