Mongolian Journal of Biological Sciences 2004 Vol. 2(1): 29-37 A Study to Detect CAM Plants in Mongolia Shagjjav Oyungerel1, Tsagaanbandi Tsendeekhuu1 & Gundsambuu Tserenkhand2 1Faculty of Biology, National University of Mongolia, Ulaanbaatar 210646, Mongolia e-mail: [email protected] 2Botany Institute, Mongolian Academy of Science, Ulaanbaatar, Mongolia Abstract In order to discover CAM plants from the Mongolian flora, four species, Orostachys spinosa (L.) C. A. Mey., O. malacophylla (Pall.) Fisch., O. thyrsiflora Fisch. and Sedum aizoon L. of Crassulaceae D.C. family were examined in terms of their leaf anatomy, photosynthesis and transpiration intensity for a 24- hour cycle. Photosynthesis by these plants has been studied using isotope-discriminate analysis (δ13C) and a special method for CAM. Transpiration was measured by the weight-method and leaf anatomy and 13 12 stomatal movement by microscopy. C/ C value of Orostachys spinosa, O. thyrsiflora C4-like (-13.44%ο,- 18.10%ο), O. malacophylla, Sedum aizoon C3-like (-25.03%ο,-26.32%ο). CAM plant characters are clearly discovered in two species Orostachys spinosa and O. malacophylla. Although differences in the acidity value cycle of Sedum aizoon in terms of a 24-hour cycle was similar to the previous two plants, stomatal movement was commonly closed during night and day showing that we need to conduct more future studies for this species. Orostachys thyrsiflora does not have CAM photosynthetic response. Key words: leaf anatomy, CAM, photosynthesis, transpiration intensity, stomatal movement Introduction Nowadays, an estimated 15-20,000 CAM species are known in 33 families in terrestrial and During the past several years drought also in aquatic plants (Black & Osmond, 2003). occurrence has increased in Mongolia, and drastic From those, 14 families grow in the Mongolian changes have taken place in climate, there has been flora including Asteraceae, Chenopodiaceae, a significant increase detected in desertification and Caryophyllaceae which are the largest families in degradation has occurred in pasturelands. For this Mongolia’s flora. reason, there is an essential need to study the eco- In crassulacean acid metabolism (CAM), the physiology of succulent and CAM (Crassulaceaen reactions of photosynthesis and CO2 uptake are Acid Metabolism) plants, which have the ability temporally separated; CO2 uptake and fixation take to keep the water in their cells, can endure dry, hot place at night, and decarboxylation and refixation conditions and grow in desert areas. These plants of the internally released CO2 occur during the day. are poorly studied in Mongolia. CAM is an adaptation primarily to minimize the CAM plants open their stomata at night and quantity of water that is lost when stomata are close them during the daytime to survive hot and opened to permit the entry of CO2. In CAM plants, dry conditions. CAM metabolism was first the stomata are opened in the cool of the night. investigated in members of the family CO2 is fixed as malic acid, which is stored in the Crassulaceae D.C. and called Crassulaceaen Acid vacuole. As malic acid accumulates, the leaf Metabolism. The Crassulaceae Family has 40 vacuoles acidify in the dark. Upon illumination, genus, 15,000 species and CAM has been recorded the stomata close, and the leaf deacidifies. The in 25 genus: Adromischus, Aeonium, Bryophyllum, malic acid is recovered from the vacuole and Cotyledon, Crassula, Cremnophila, Diamorpha, undergoes decarboxylation. The CO2 that is Dudleya, Echeveria, Graptopetalum, Greenovia, released is prevented from escaping by stomatal Hasseanthus, Hylotelephium, Kalanchoe, closure and is assimilated via the Calvin cycle using Lenophyllum, Monanthes, Nananthus, photochemically generated ATP and NADPH Pachyphytum, Parvisedum, Rochea, Sedum, (Hatch & Boardman, 1981). Sempervivum, Tylecodon, Umbilicus, Villadia (″http://biodiversity.uno.edu/delta). http://dx.doi.org/10.22353/mjbs.2004.02.04 29 Sh. Oyungerel et al. Materials Methods We have selected the following 4 species of Our experiments were conducted in 2003 during Crassulaceae. Orostachys spinosa, O. a field expedition, mainly in forest steppe and semi- malacophylla, O. thyrsiflora, Sedum aizoon (Table desert areas in the following places: Monostoin am 1). They are all leaf succulent plants and the leaf’s (49°19′N, 106 °35′Ε) of Shariin gol somon, water content is as follows: Orostachys spinosa Darkhan uul province and near Jinst Khairhan (96.17%), O.malacophylla (95.32%), O.thyrsiflora mountain (44°32′N, 99°15′Ε) of Shine Jinst somon, (89.56%), Sedum aizoon (91.38%). The plants were Bayankhongor province. growing in their natural habitats. Plants were taken Photosynthetic types were determined from from near Monostoi am, Orostachys spinosa, O. δ13C fractionation, δ13C analysis was performed malacophylla and Sedum aizoon grow in steep, with a United States National Bureau of Standards rocky places on the southern slopes of mountains bicarbonate sample as a standard. Plant and forest edge. O .thyrsiflora grows in rocky soil photosynthetic tissue samples were field-collected in desert-steppe region. in natural habitats, sun dried and transported to the Table 1. Morphology, distribution, habitat and utilization of succulents* Distribution in botanico- Description geographical regions of Habitat Mongolia Species Utilization 5-20cm high, leaves in rosette crowded, Khubsugul, Khentei, Khan- Stony and oblong, apical spine 2-4mm, stem leaves gai, Mongol-Daurian, Middle debris slopes, linear to oblanceolate, 1-3cm, acuminate, khalkha, East Mongolia, rocks, water- flowers in raceme or spike, sepals red Khobdo, Mongol Altai, side pebbles, variegate, anthers yellow. Flowers Depression of Great lakes sayrs greenish-yellow, solitary sessile or on (Uvs Nuur), Valley of Lakes, cattlesmall very short pedicels. East Gobi, Gobi Altai Medicine, food for Orostachys spinosa 10-30cm high, rosette 4-15cm, leaves Khubsugul, Khentei, Khan- Debris and oblong to elliptic, entire, without apical gai, Mongol-Daurian, Great stony slopes of spine, flowers in raceme, bracts Khingan, Middle khalkha, hills and spathulate-ovate, sepals oblong, petals East Mongolia mountains, ovate-oblong, 4-6mm long. Flowers rocks, dry stone yellow. screes, sandy O. malacophylla steppes Food for small cattle 10-20cm high, leaves in rosette green, Khentei, Khangai, Mongol- Rocks, debris imbricate, linear-lanceolate, 1-2mm, Daurian, Great Khingan, and stony shortly mucronate, stem leaves oblong, Khobdo, Mongolian Altai slopes, screes, 4-7mm, flowers in raceme, sepals Middle khalkha, East waterside sands triangular-ovate, anthers dark purple. Mongolia, Valley of Lakes, and pebbles Flowers white or pinkish, by several on Gobi Altai, Dzungarian Gobi, O. thyrsiflora. elongated peduncles. Transaltai Gobi Food for cattle small Flowers yellow; rhizome short, mainly Khubsugul, Khentei, Khan- Rocks, stony thick, irregularly accrecsent, giving rise gai, Mongol-Daurian, Great mountain to solitary or few straight simple and Khingan, Middle khalkha, slopes, screes, aizoon firm, annually dying off flowering stems East Mongolia, Khobdo, sandy-pebble 15-40 cm high. Leaves lanceolate, Mongolian Altai, Valley of river banks in sometimes sublinear, somewhat acute, Lakes, Gobi Altai forest and forest Medicine Sedum irregularly serrate-dentate, up to 3-5 (7) steppe belts *(Ulziikhutag, 1985; Gubanov, 1996; Grubov, 2001) 30 CAM plants in Mongolia USA for δ13C analysis. Microscopic studies of leaf Table 2. 13C/12C value of species anatomy and daily stomatal movement. For δο13C% detecting CAM plant were determined daily pH, Species C -like C - like stomatal movement and transpiration intensity. We 3 4 Orostachys spinosa -13.44 did our research in the natural habitats, hourly and Orostachys malacophylla -25.03 using fresh undamaged leaves, including three Orosotachys thyrsiflora -18.10 plants with three repeats. We did research usually Sedum aizoon -26.32 on sunny, non-rainy and non-windy days. To detect acidity we used pH indicator (paper) and titrable acid. Results of these studies However, when some CAM plants are well watered, they switch to C mode by opening their correspond with each other so we show acidity by 3 stomata and fixing CO during the day via rubisco. pH value. We used fast weight-method (Vicktorov, 2 1969): first choose an undamaged leaf positioned Under these conditions the isotope composition shifts more toward that of C plants. Thus 13C/12C at mid-plant height. Take the weight just after 3 isolating and after 3 minutes. During this time the values for CAM plants reflect how much carbon is fixed via the C pathway versus the C pathway leaf should be kept in a natural environment. Then 3 4 calculate transpiration intensity by the following (Hatch & Boardman, 1981). formula: 1. For detecting CAM plants T =(a-d)*20/a We conducted experiments on the following i parameters over a 24-hour cycle at 2 and 3 hour Ti – transpiration intensity a – first weight intervals to examine whether these plants show d – weight after 3 minutes CAM photosynthetic responses. The examined parameters are: acidity and pH values, stomatal We studied stomatal movement by using the movement, transpiration intensity. pattern method by Molotkovskii (Vicktorov, 1969). First we prepared an ointment by dissolving a a. Orostachys spinosa photography film in acetone. Then paint the leaf pH value: pH value for 24-hour cycle was surface with the ointment. When the acetone is studied at 2-hourly intervals by testing pH value steamed this