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A Study to Detect CAM Plants in Mongolia

Shagjjav Oyungerel1, Tsagaanbandi Tsendeekhuu1 & Gundsambuu Tserenkhand2
1Faculty of Biology, National University of Mongolia, Ulaanbaatar 210646, Mongolia
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 (d13C) and a special method for CAM. Transpiration was measured by the weight-method and leaf anatomy and stomatal movement by microscopy. 13C/12C value of Orostachys spinosa, O. thyrsiflora C4-like (-13.44%o,- 18.10%o), O. malacophylla, Sedum aizoon C3-like (-25.03%o,-26.32%o). 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.

Keyword: leaf anatomy,CAM,photosynthesis,transpiration intensity,stomatal movement

Introduction

During the past several years drought occurrence has increased in Mongolia, and drastic changes have taken place in climate, there has been
a significant increase detected in desertification and degradation has occurred in pasturelands. For this reason, there is an essential need to study the ecophysiology of succulent and CAM (Crassulaceaen Acid Metabolism) plants, which have the ability to keep the water in their cells, can endure dry, hot conditions and grow in desert areas. These plants are poorly studied in Mongolia. CAM plants open their stomata at night and close them during the daytime to survive hot and dry conditions. CAM metabolism was first investigated in members of the family Crassulaceae D.C. and called Crassulaceaen Acid Metabolism. The Crassulaceae Family has 40 genus, 15,000 species and CAM has been recorded in 25 genus: Adromischus, Aeonium, Bryophyllum, Cotyledon, Crassula, Cremnophila, Diamorpha, Dudleya, Echeveria, Graptopetalum, Greenovia, Hasseanthus, Hylotelephium, Kalanchoe, Lenophyllum, Monanthes, Nananthus, Pachyphytum, Parvisedum, Rochea, Sedum, Sempervivum, Tylecodon, Umbilicus, Villadia (²http://biodiversity.uno.edu/delta). Nowadays, an estimated 15-20,000 CAM species are known in 33 families in terrestrial and also in aquatic plants (Black & Osmond, 2003). From those, 14 families grow in the Mongolian flora including Asteraceae, Chenopodiaceae, Caryophyllaceae which are the largest families in Mongolia’s flora. In crassulacean acid metabolism (CAM), the reactions of photosynthesis and CO2 uptake are temporally separated; CO2 uptake and fixation take place at night, and decarboxylation and refixation
of the internally released CO2 occur during the day. CAM is an adaptation primarily to minimize the quantity of water that is lost when stomata are
opened to permit the entry of CO2. In CAM plants, the stomata are opened in the cool of the night. CO2 is fixed as malic acid, which is stored in the vacuole. As malic acid accumulates, the leaf vacuoles acidify in the dark. Upon illumination, the stomata close, and the leaf deacidifies. The malic acid is recovered from the vacuole and undergoes decarboxylation. The CO2 that is released is prevented from escaping by stomatal closure and is assimilated via the Calvin cycle using photochemically generated ATP and NADPH (Hatch & Boardman, 1981).

Material and Methods

Materials: We have selected the following 4 species of Crassulaceae. Orostachys spinosa, O. malacophylla, O. thyrsiflora, Sedum aizoon (Table 1). They are all leaf succulent plants and the leaf’s water content is as follows: Orostachys spinosa (96.17%), O.malacophylla (95.32%), O.thyrsiflora (89.56%), Sedum aizoon (91.38%). The plants were growing in their natural habitats. Plants were taken from near Monostoi am, Orostachys spinosa, O. malacophylla and Sedum aizoon grow in steep, rocky places on the southern slopes of mountains and forest edge. O .thyrsiflora grows in rocky soil
in desert-steppe region.
Methods: Our experiments were conducted in 2003 during a field expedition, mainly in forest steppe and semidesert areas in the following places: Monostoin am (49°19¢N, 106 °35¢E) of Shariin gol somon, Darkhan uul province and near Jinst Khairhan mountain (44°32¢N, 99°15¢E) of Shine Jinst somon, Bayankhongor province. Photosynthetic types were determined from d13C fractionation, d13C analysis was performed with a United States National Bureau of Standards bicarbonate sample as a standard.

Plant photosynthetic tissue samples were field-collected in natural habitats, sun dried and transported to the USA for d13C analysis. Microscopic studies of leaf anatomy and daily stomatal movement. For detecting CAM plant were determined daily pH, stomatal movement and transpiration intensity. We did our research in the natural habitats, hourly and using fresh undamaged leaves, including three plants with three repeats. We did research usually on sunny, non-rainy and non-windy days. To detect acidity we used pH indicator (paper) and titrable acid. Results of these studies correspond with each other so we show acidity by pH value. We used fast weight-method (Vicktorov, 1969): first choose an undamaged leaf positioned at mid-plant height. Take the weight just after isolating and  after 3 minutes. During this time the leaf should be kept in a natural environment. Then calculate transpiration intensity by the following
formula:
Ti=(a-d)*20/a
Ti – transpiration intensity
a – first weight
d – weight after 3 minutes
We studied stomatal movement by using the pattern method by Molotkovskii (Vicktorov, 1969). First we prepared an ointment by dissolving a photography film in acetone. Then paint the leaf surface with the ointment. When the acetone is steamed this layer becomes solid, revealing the stomatal pattern and we can count and measure stomatal movement by viewing it through a microscope. This method does not influence opening or closure of stomata. To detect stomatal movement of the leaf we counted the stomata three times every two hours during the experiment under the same magnification after which we calculated the percentage ratio of open and closed stomata.

Result

1. d13C-analysis As a result of d13C analysis Orostachys spinosa and Orosotachys thyrsiflora were found to have C4 photosynthesis; Orostachys malacophylla and Sedum aizoon have C3 photosynthesis (Table 2). C3 plants have d13C of -35-(30-25)-20%o; C4 plants have d13C -20-(15-10)-5%o (Taiz Zeiger 1998). CAM plants can have d13C values that are intermediate between those of C3 and C4 plants. In CAM plants that fix CO2 at night via PEP carboxylase, d13C is similar to that of C4 plants.

However, when some CAM plants are well watered, they switch to C3 mode by opening their stomata and fixing CO2 during the day via rubisco. Under these conditions the isotope composition shifts more toward that of C3 plants. Thus 13C/12C values for CAM plants reflect how much carbon
is fixed via the C3 pathway versus the C4 pathway (Hatch & Boardman, 1981).
1. For detecting CAM plants
We conducted experiments on the following parameters over a 24-hour cycle at 2 and 3 hour intervals to examine whether these plants show CAM photosynthetic responses. The examined parameters are: acidity and pH values, stomatal movement, transpiration intensity.
a. Orostachys spinosa
pH value: pH value for 24-hour cycle was studied at 2-hourly intervals by testing pH value in the leaf samples. The acidity value was relatively higher (pH = 4.5-4.75) during the night from 1am to 9am, and decreased (pH = 5.0-5.5) in the morning 11am, increased in the evening 11pm and reached its maximum value (pH = 4.5) in the morning from 7am to 9am (Fig. 1). Stomatal movement: Over a 24-hour cycle, it was detected that a stomata was fully closed from 5am in the morning to 5pm in the evening. From 7pm in evening the numbers of opening and closing stomata was equal. From 11pm to 3am, the number of open stomata increased and the stomata was fully opened at 11pm. (Fig. 1).
Transpiration intensity: The cycle of transpiration intensity generally matched the movement of stomata (Fig. 2). The transpiration intensity was
highest, 0.34 g/mg per hour, when stomata were fully open in the night 11pm. There was no transpiration when stomata were fully closed at 9am. Although there is a detectable value of transpiration intensity during the daytime i.e. 1-5pm (0.06-0.11 g/mg per hour), it is not stomata transpiration but cuticle transpiration.
Since transpiration intensity was relatively small during the daytime, leaf stomata of Orostachys spinosa were open during the night, and pH value was higher in the night than during the day, it suggests that this plant has CAM photosynthesis.
b. Orostachys malacophylla
pH value: Acidity value increased (pH = 4.5- 5.25) between 1am and 11am in the morning, and decreased (pH =4.7-5.75) from 1pm to 11pm (Fig.
3). Stomatal movement: The stomata were fully opened at 3am, while 67-100% closed from 5am to 9pm. The number of open stomata increased in the evening 11pm (Fig. 3).
Transpiration intensity: The cycle of transpiration intensity also matches the movement of stomata (Fig. 4).
From 11pm to 3am, there was higher transpiration intensity, 0.13 - 0.30 g/mg per hour, while rather low transpiration (0.02-0.08 g/ mg per hour) was found during the rest of the time when 80-100% of stomata were closed. The results for Orostachys malacophylla were similar to Orostachys spinosa suggesting that this plant also has CAM photosynthesis.
c. Orostachys thyrsiflora
pH value: There was an unremarkable difference (pH = 4.5-5.5) between day and night when the cycle of acidity was considered at the 3- hourly interval (Fig. 5). Stomatal movement: Majority of stomatal cells (67%) were open in the morning 10am; closed numbers were higher (57-75%) during the remaining 24-hours (Fig. 5).
Transpiration intensity: The cycle of transpiration intensity matched the movement of stomata. The transpiration intensity was higher, 0.34 g/mg per hour, at 10am, when more stomata were open. Lower intensity, 0.06-0.11 g/mg per hour, was found during the rest of the time when a large percentage of stomata were closed (Fig. 5).
The result that there was no difference in acid variation during day and night and no open stomata during the night time suggest that Orosotachys
thyrsiflora does not have CAM photosynthetic response.
d. Sedum aizoon
pH value: Acid value was relatively high (4.75- 5.0) from 3am to 9am, but decreased from 11am to 9pm (5.25-5.5) and then increased again at 11pm
and 1am (Fig. 6).
Stomatal movement: The stomatal cells were more often closed than open apart from at 5pm (Fig. 7). Transpiration intensity: The transpiration intensity was 0-0.13 g/mg per hour, increases and decreases generally matched the movement of stomatal cells. (Fig. 7).
The results for Sedum aizoon suggest that it has the same regime as a CAM plant, since the acidity value was high at night and low in the day with
the majority of stomatal cells remaining closed for most of the time. Opening and closing times of the stomatal cells corresponded to the increase and decrease of transpiration intensity for this plant.
3. Leaf anatomy
a) Orostachys spinosa
(Fig. 8)
Leaf anatomy has a homogenous structure. Mesophyll composed of multi- layered homogenous spongy mesophyll. Anisocytic stomata evenly distributed in the surface of the leaf and located in same level with epidermal cells. Epidermal cells are evenly small, oval in cross section.
b) Orostachys malacophylla (Fig. 9)
Epidermal cell wall slightly undulated and thickened. Stomata are diacytic type. Stomatal air space is bigger. Cells around the bunds are relatively large and some of them store water.
c) Orostachys thyrsiflora (Fig. 10)
Epidermal cell wall thickened unevenly. Stomata are anisocytic type. Air space is smaller. Bunds well-developed and neighboring cells store a water. Mesophyll cells are smaller and composed of more layers. Cross section of leaf is semi-circular
d) Sedum aizoon (Fig. 11)
Leaf has homogenous structure. Mesophyll tissue stores water besides photosynthesis. Mesophyll cells are big and have a thin wall. Epidermal cells are big and outer walls are thick. Evenly distributed anisocytic stomata. Bunds are collateral.

Discussion

The large group of plants with powerful water conserving types of physiology and biochemistry called Crassulacean Acid Metabolism (CAM) are absent from deserts of Central Asia, but present in other deserts of the world. The deserts of America, Africa and Australia contain large numbers of huge, even tree-like CAM plants such as cactus, Agave, Aloe, and Euphorbs. In fact, for a decade we have searched in Central Asia for CAM plants, but they are absent. (Black et al., 2003). However, we discovered CAM in one native Asian genus, Orostachys in our experiments. CAM plant characters are clearly discovered in the following two species Orostachys spinosa and O. malacophylla. The three main characteristics are higher night and lower daytime acidity values, leaf stomata are open during the night, and the higher transpiration intensity at night than during the
daytime. Although the cycle of acidity in Sedum aizoon was the same as the previous two plant species, the stomatal movement that was commonly closed during both night and day times suggests that we need to conduct further studies on this species.
In contrast to CAM plants, the results for O. thyrsiflora which has similar acidity value during day and night, and following the higher number of open cells in the daytime same as other plants, the increases of transpiration intensity in daytime was high, but low at night. However d13C%o value of
O. thyrsiflora was similar with C4 plants but as a result of leaf anatomy O. thyrsiflora hasn¢t got Kranz anatomy. Therefore we need to conduct further studies on this species. Maybe it is CAM or C4 plant. Some C4 species are with and without a Kranz type of leaf anatomy (Pyankov et al., 1997). Certainly with so many land and water CAM species, such strong environmental control traits, and being both constitutive and inducible, CAM remains with many useful and informative facets to be discovered! For example, two new types of photosynthesis that are intermediate between CAM and C4 (Black & Osmond, 2003).
During early d13C studies it became clear that CAM photosynthesis was both inducible and constitutive. Numerous species of plants express
CAM throughout their autotrophic lifetime. But another difficult problem in CAM photosynthesis is that, with a few plants, the stage of plant development affects CAM expression in green tissues, in combination with environmental stresses. For example, in nature Mesembryanthenum crystallinum will express C3 photosynthesis when young and slowly shift to CAM as it matures and is water stressed (Black & Osmond, 2003). Mesembryanthenum crystallinum is succulent undershrub and grows in Karru¢s subdesert of South Africa (Pukareka, 1982). Prior to this CAM was recorded in Sedum genus, but CAM has not been recorded in Orostachys genus of Crassulaceae family (http:// biodiversity.uno.edu/delta). Thus, a new genus (Orostachys) added to CAM by our study. Now, Sedum aizoon is not evident whether it is CAM or not and we need to conduct further studies on this species. An unusual night fixation of CO2 via CAM has been detected that allows these naturally occurring plants to live in cold, higher altitude from 600 to 2900 m asl) and rocky desert environments. We found that CAM occurred in succulent plant genus Orostachys in Mongolia, where it is used as food for pastoral livestock and in herbal medicine. Subsequently we found there are about 30 species of Orostachys in the world, they are widely adapted to survival and grow in Asian mountain deserts. The species of this genus are in Russia, Tibet, Japan, Korea, China, Mongolia, Siberia, Sakhalin, Kazakhstan and other areas of Central Asia. Also Orostachys was cultivated in gardens and houses in USA (Georgia), Germany (Halle), Belgium and Japan. Only four species of Orostachys have been recorded in the Mongolian flora (Gubanov, 1996). They are represented phytogeographically as the North Central Asian rock steppe element, part of which extends into the Siberian taiga and in East Asia. Occasionally Orostachys spinosa and Chamaerhodos altaica have been found on bare rock and on cobbled and skeletal soils in the eastern Hangai (Hilbig, 1995). Orostachys spinosa remains green under snow cover during the Mongolian winter. Mongolia has an extreme continental climate with extremes of cold and heat and very low precipitation. The absolute temperatures vary from -50°C in winter to +40°C in summer (Pyankov et al., 2000).
Therefore, it seems to be the most cold adapted CAM plant known, its special environmental adaptation mechanisms remain to be elucidated. It is one of the important goals of our future study.

Acknowledgement

The research was supported by the Asian Research Center (ARC) at the NUM. We wish to thank to Prof. Clanton C. Black of Georgia
University, USA. Thanks are due to anonymous reviewers for their critical reading of the manuscript.

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