Annals of RSCB Vol. XVIII, Issue 1/2013

ESEM AND EDAX OBSERVATIONS ON LEAF AND STEM EPIDERMAL STRUCTURES (STOMATA AND SALT GLANDS) IN LIMONIUM GMELINII (WILLD.) KUNTZE Iulia-Natalia Daraban 1, C. V. Mihali 1, Violeta Turcus 2, A. Ardelean 2, G.-G. Arsene 2,3 1 LIFE SCIENCES INSTITUTE WESTERN UNIVERSITY VASILE GOLDIS ARAD; 2 VASILE GOLDIS WESTERN UNIVERSITY OF ARAD; 3 BANAT S UNIVERSITY AGRICULTURAL SCIENCES AND VETERINARY MEDICINE TIMISOARA, FACULTY OF AGRICULTURE Summary Plants sampled in a Limonium gmelinii population in Vărsand (Arad, Romania), from a permanent salty meadow are examinated in ESEM and EDAX. The observed structure are the salt glands and the stomata on leaf and stem epidermis. Specimen were obtained from 10 individual plants. Measurements concern the area of salt glands, their density in leaves epidermis, the longitudinal and transversal dimensions of stomata, and the distance between the salt glands and the nearest stomata. A higher salt glands area was found in leaves adaxial epidermis, compared to the abaxial epidermis, but the limits of variation are alike. The stomata are longer in stem epidermis, but their mean width is lower than in leaves epidermis. The main chemical element, revealed by EDAX observation, in salt glands excreta is calcium. Key words: Limonium gmelinii, ESEM, EDAX, stomata, salt glands. mihaliciprian@yahoo.com Introduction Genus Limonium comprises annuals and perennials (about 300 species Tse- Hsiang & Kamelin, 1996) with sexual and / or asexual (apomixis) reproduction (Róis et al., 2012). The areal of Limonium gmelinii goes from Europe to Mongolia and China. From an ecological point of view, this species is a halophyte with salt-excreting glands, classified in different halophytes categories according to different authors (e.g. regretohalophyte or crino-halophyte; the limits of separating halophytes in subtypes are discussed by Grigore et al., 2010). Limonium gmelinii is a very potassiophilic species (Breckle, 2004) and the salt glands start to excrete NaCl only after a distinct salinity level in the leaf is reached (Wiehe, 1986, in Breckle, 2004). Limonium gmelinii was studied from a biochemical point of view (Korul kina et al., (a), (b), 2004; Zhusupova & Abil kaeva, 2006 etc); other authors tested the antioxidant capacity of L. gmelinii extracts (Smirnova et al., 2009). We published a review of medicinal uses (Dărăban et al., 2013) of this plant. In Romania, Motiu et al. (1987) studied the vegetative organs in light microscopy, and there is a similar study published by Kazach researchers (Aydosova et al., 2012). Leaves of L. gmelini, are bifacial, with anisocytic stomata, at the same level as epidermal cells (Zhou et al., 2007, SEM observation); the salt glands and stomata are also distributed on both leaf sides (Zhou et al., 2006). The salt glands of Limonium gmelini are composed of 16 glandular cells, overlying 4 collecting cells that are connected to mesophyll cells by plasmodesmata. A layer of cutin surrounds the collecting cells except at pore interruptions (Dickison, 2000). In this study we present ESEM (Environmental Scanning Electron Microscopy) observations with measurements of epidermal features and EDAX (Energy Dispersive X-ray Analysis) observations on salt glands excreta and normal epidermal cells. 123

Materials and methods Leaves and stem were sampled from plants growing in Vărsand (Arad department), in August 2012. We used leaves from the medium part of rosettes and fragments of stems cut from their midlength. As parameters, we measured: - the salt glands area (fig. 1., area of upper parts of salt glands, i.e. the 4 secretory cells and the salient cup of the gland); - the length of stomatal cells (fig. 2., the longitudinal dimension) - the width of stomatal cells (fig. 2., the transversal dimension); - the salt gland density per mm 2 ; - the distance between the limits of the salt gland and the limits of surrounding stomata (µm, fig. 3.). The samples for ESEM investigations were mounted on conductive copper or aluminium carriers, with the help of both sides adhesive carbon discs, over which a 0,45 µm Millipore filter is added. In this phase, the sample orientation on the carrier is very important, considering the limited possibilities of inclining the sample within the microscope, so that interest area will be directly exposed to the scanning electron beam. Though, the samples mounting was realized with the help of a stereomicroscope, facilitating their arrangement on a single carrier. For the mounting of specimens with high water content, the same combination of mounting reagents is used as in transmission electron microscopy, as following: pre-mounting with glutaraldehyde 2,7 % within 0,1 M phosphate buffer, ph 7,4, for 60-90 min at 4 C; clearing the excess of mounting reagent by passing the sample through 0,15 M phosphate buffer, ph 7,4, three consecutive baths, of 60 min. each, the fourth being held overnight, all at 4 C; post-mounting with osmic acid, (OsO4) in 0,15 M phosphate buffer, ph 7,4, for 75-90 min., at 4 C. The samples examination is made after they are mounted on the cooling stage and the Peltier plate. Initially the cooling stage is introduced within the scanning electron microscope and cooled at 3 C for 15 min. The sample is mounted on the cooling stage, and after is closed within the examination chamber. The examination is done at 3 C temperature, 100% relative humidity and 910 Pa pressure. The stage with the sample is lifted at 5 mm working distance of the polar piece of the microscope, reason why general/entire piece images cannot be captured. The work was made with spot 5 and an acceleration tension of 10 kv, with the GSED detector (Gaseous Secondary Electron Detector). To avoid the dehydration of the sample, the examination time has to be around 10-30 min. The samples don`t have to be coated. The examination is made with a Fei Quanta 250 scanning electron microscope. For EDAX, all samples were examined with gold sputtering on their surface, they also were mounted on conductive aluminium pin stub specimen using adhesive carbon discs on both sides. EDAX analysis parameters were HV mode, 15kV, ETD, EDAX, spot 5, WD 5 mm. Determination of chemical elements was performed using abaxial epidermis (a fresh sample of normal epidermal cells and a naturally dried sample of excreted salt corpuscles). Preliminary, both samples were metalized with gold, searching for carbon, oxygen, magnesium, gold (from the SEM sample preparation), sulphur, chlorine, potassium, and calcium (on fresh sample), and respectively carbon, oxygen, magnesium, gold (from the SEM sample preparation), sodium, chlorine, potassium, and calcium (on naturally dried sample). 124

Fig. 1. Example of the measurement of salt gland area. Fig. 2. The two transversal dimensions of stomata (leaf, adaxial epidermis) Fig. 3. Example of a measurement of the distance between the salt gland and the surrounding stomata (leaf, adaxial epidermis). Results and Discussions A synthesis of performed measurement is presented in tab. 1. We noticed no significant differences in the general aspect of the leaf adaxial and abaxial epidermis, except the more obvious radial disposal of cells surrounding the salt gland (fig. 4., 5.) in the abaxial epidermis. The area of the 4 superior cells of a salt gland (the entire salt gland is made by 16 or 20 cells Breckle, 2004) appears to be higher on adaxial epidermis. 125

A B C cs ce D cst îc ca Fig. 4. Adaxial fresh leaf epidermis (SEM images): A, B general aspects; C detailed image, ce cup s edge, cs secretory cells, îc cuticular belt thickenings, ca annex cells, cst stomatic cells; D detail containing a calcium corpuscle. 126

A B îc cs ce ca cst C cs D cst ce îc ca Fig. 5. Abaxial fresh leaf epidermis (SEM images): A general aspect; B, C, D details: cs secretory cells, ce cup s edge, îc - cuticular belt thickenings, ca annex cells, cst stomatic cells. Table 1. The values of measured parameters (30 counts for each parameter) Parameter Average Min. Max. Leaf: Adaxial epidermis: Salt glands areas (µm 2 ) 976.75 733.98 1308.67 Length of stomatal cels (µm) 30.47 26.39 35.24 Stomata width (µm) 25.07 17.97 31.57 Salt glands density (per cm 2 ) 756 632 843 Abaxial epidermis: Salt glands areas (µm 2 ) 901.34 693.23 1267.78 Length of stomatal cels (µm) 29.34 24.45 36.30 Stomata width (µm) 23.96 17.93 30.48 Salt glands density (per cm 2 ) 668 512 701 Distance between the limits of the salt gland and 58.28 46.77 77.26 the limits of surrounding stomata (µm) Stem: Salt glands areas (µm 2 ) 738.18 517.13 983.45 Length of stomatal cels (µm) 34.57 40.56 Stomata width (µm) 17.66 15.67 19.57 The presence of the 4 pores per salt gland (fig. 6) is a constant feature. In active glands, excreted salt formations may covered completely the cup of the gland (fig. 8), and salt corpuscles are sometimes visible on the normal epidermal cells surface (fig. 4. D). The highest average length of stomata was found in stem 127

Annals of RSCB Vol. XVIII, Issue 1/2013 epidermis (34.57 µm), whilst the same parameter is contiguous in leaf both epidermis (tab. 1). Transversal dimension (width) of stomata is lower in stem epidermis, due, the most probably, to the general orientation of all epidermal cells and to the tissular tensions issued from the growing process (fig. 8); the same tensions may explain the quadrangular shape of the salt glands (fig. 9). Average salt glands density in leaf epidermis is 756 / cm2 (adaxial epidermis) and respectively 668 / cm2; Ruhland (1915, in Grigore, 2008) reports close values: 722 and 644. The average distance between the external walls of the salt glands cells and the closest stomata, in abaxial epidermis has a mean value of 58.28 µm. Fig. 6. Salt gland (leaf, adaxial epidermis); the 4 pores are clearly visible. Fig. 7. Salt gland (leaf, abaxial epidermis); the pores are covered with excreted salt corpuscles. Fig. 8. Stem epidermis with stomata. Fig. 9. Salt glands in stem epidermis. EDAX analysis shows the presence of the same chemical elements in both type samples, except the sodium, found only in excreted corpuscles (fig. 10), and the sulphur, found on normal epidermal cells (fig. 11). Huge amounts of calcium were revealed in excreted corpuscles. 128

Fig. 10. EDAX smart-maps: excreted salt corpuscles on abaxial epidermis (naturally dried leaf sample). Fig. 11. EDAX smart-maps: normal epidermis cells, abaxial epidermis (fresh leaf sample). Conclusions Plants from the Vărşand population show differences in epidermis structure according to the examinated organ. The area of salt glands is slightly higher in adaxial leaf epidermis compared to the abaxial epidermis (976.75 µm 2, and 901.34 µm 2, respectively). Stem stomata are longer compared to the leaves stomata, but their average width is lower. Salt glands in stem epidermis show a quadrangular shape. Acknowledgement This work was supported by Structural Funds POSDRU/CPP107/DMI 1.5/S/77082 Burse doctorale de pregătire References Aydosova, S.S., Zhussupova, G.E., Akhtayeva, N.Z., Gadetskaya, A.V., Abilov, Z.A., Macro- 129 EDAX analysis attests the presence of calcium in salt excretory glands products. The epidermis characteristics could be used as a complementary tool in investigating differences in sub-specific taxa and populations living in different soil conditions. Further researches in genetics are needed in order to evaluate these differences and / or the ecological plasticity in Limonium gmelinii populations. ecoeconomică şi bioeconomică complexă pentru siguranţa şi securitatea alimentelor şi furajelor din ecosisteme antropice and microscopy of upper parts from Limonium gmelinii genus plants, International Journal of Biology and Chemistry, 4, pp. 3-6, 2012.

Breckle, S.-W., - Salinity, Halophytes and Salt- Affected Natural Ecosystems, in: Läuchli, A., Lütge, U. (editors), 2004 Salinity: Environment Plants Molecules, Kluwer Academic Publishers, New York, Boston, Dordrecht, London, Moscow, pp. 53-77, 2004. Dărăban, I.-N., Arsene, G.-G., Turcus, V., Ardelean, A., Assessment on bioeconomical potential for medicinal plants in salty meadows from the Aradului Plain (W. Romania), Studia Universitatis Vasile Goldis, Seria Stiintele vietii, Vol. 23, issue 1, pp. 83-90, 2013. Dickison, W.C., Integrative Plant Anatomy, Academic Press, San Diego, California, p. 307, 2000. Grigore, M.-N., Introducere în halofitologie. Elemente de anatomie integrativă, Ed. PIM, Iasi, 2008. Grigore, M.-N., Toma, C., Boșcaiu, M., Dealing with halophytes: an old problem, the same continuous exciting challenge, Anale știinșifice ale Universitătii Al. I. Cuza Iași, Tomul LVI, fasc. 1, s IIa. Biologie vegetală, pp. 21-32, 2010. Korul kina, L.M., Shul ts, E.E., Zhusupova, G.E., Abilov, Z.A., Erzhanov, K.B., Cahudri, M.I., (a), Biologically active compounds from Limonium Gmelinii and L. Popovii, I., Chemistry of Natural Compounds, 40 (5), pp. 465-471, 2004. Korul kina, L.M., Zhusupova, G.E., Shul ts, E.E., Erzhanov, K.B., (b), Fatty-acid composition of two Limonium plant species, Chemistry of Natural Compounds, 40 (5), pp. 417-419, 2004. Moșiu, T., Toma, C., Tiron, A., Nită, M., Contributii la cunoasterea structurii organelor vegetative la Limonium gmelini (Willd.) O. Ktze, Analele Stiintifice ale Universitătii Al. I. Cuza din Iași, Tomul XXXIII, s. II-a, Biologie, pp. 11-16, 1987. Róis, A.S., Teixeira, G., Sharbel, T.F., Fuchs, J., Martins, S., Espirito-Santo, D., Caperta, A.D., Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes, Sex Plant Reprod, 25, pp. 305-318, 2012. Smirnova, G.V., Vysochina, G.I., Muzyka, N.G., Samoilova, Z.Y., Kukushkina, T.A., Oktyabr skii, O.N., The Antioxidant Characteristics of Medicinal Plant Extracts 130 from Western Siberia, Applied Biochemistry and Microbiology, 45 (6), pp. 638-641, 2009. Tse-Hsiang, P., Kamelin, R.V., Plumbaginaceae, in: Flora of China, 15, pp. 190-204,2009, at URL: http://flora.huh.harvard.edu/china/mss/volume 15/Plumbaginaceae.published.pdf, accessed on April, 14 th, 2013 Zhou, L.-L., Liu, P., Lu, J.-H., A SEM observation of the salt-secreting structure of leaves in four species of Limonium, Bulletin of Botanical Research, 26 (6), pp. 667-671, 2006. (abstract) Zhou, L.-L., Ping, L., Wang, J., Nutritive Organs Anatomical Structure of Two Species of Limonium in Xinjiang, Acta Botanica Boreali-Occidentalia Sinica, 6, pp. 1127-1133, 2007. Zhusupova, G.E., Abil kaeva, S.A., Flavanes from Limonium gmelinii. II., Chemistry of Natural Compounds, 42 (1), pp. 112-113, 2006.