Waxes from the Mat Sedge-Cyperus pangorei Rottb.

MATERIALS AND METHODS

C. pangorei plants were collected from the wild along rivers of Tirunelveli District., Tamil Nadu, India and maintained in the department nursery at near natural growth conditions. The waxes were extracted from the surface of the culms and three regions were selected for wax analysis based on light microscopic examination for deposition of wax, viz., the lower most 2 inches of the culm, the middle 2 inch and the topmost 2 inch of the culm.

Wax extraction and analysis: The cuticular components were extracted in chloroform as described by Rhee et al. (1998). The extracted wax samples were characterized by GC-MS (Gas Chromatography-Mass Spectrometry) analysis at SITRA (South India Textile Research Association) laboratories, Coimbatore. The lyophilized waxes were analysed using Fison 8000 series Gas chromatography with Fison MD 800 Mass Selective Detector. The column used was DBMS (30-meter length and 0.25 μm diameter). The carrier gas was Helium with a flow rate of 1.5 m min^-1. The injector temperature was 270°C with the transfer line at 175°C. Splitless mode was used with an oven program at 50°C, which was held for 2 min and steadily increased to 130°C at the rate of 9°C min^-1 and then to 280°C with a steady increase of 4°C min^-1. This temperature was held for 5 min. Compounds were identified with mass spectra of NIST95 database. For MS the conditions were full scan mode with a mass range 50-500 m/z and EI positive, ionization mode was used (Rhee et al., 1998). The several components obtained were grouped into major classes using their chemical formula and the percentage of each class was calculated by taking average and the results were tabulated.

Scanning electron microscopy: Scanning electron microscopic pictures were taken from all the three regions of the culms. The plant material was mounted on aluminium holders, sputter coated with gold-palladium (Bal-Tec SCD005 Sputter Coater; 25 mA, 300 sec; Balzers, Switzerland) and examined by SEM (Zeiss DSM 962, 15 kV; Zeiss, Oberkochen, Germany). The sputtering conditions, depositing approximately 20 nm of the alloy on the samples, were optimized for the acceleration voltage used in the SEM (Reidel et al., 2003). Simple statistical analysis like Students‘t’ test and ANOVA were performed to interpret the results and their significance.

RESULTS AND DISCUSSION

The chemical composition and physical properties of the cuticle and classification of epicuticular waxes has remained under investigation by several biologists and chemists that has resulted in fascinating significant findings. The present study on the chemical composition and morphology of epicuticular waxes of Cyperus pangorei are first of its kind in sedges and reveal several wax entities and micro morphologies when observed under SEM.

Chemical composition of epicuticular waxes: GC-MS analyses of cuticular waxes revealed that C. pangorei culms contain several aliphatic waxes and some particularly abundant major compounds given in Table 1. The kind and quantity of waxes differ from region to region. The major wax classes include alkyl esters, n-alkanes, fatty acids and primary alcohols, however, the most abundant compounds are heptacosanoic acid methyl ester, dodecatrien-1-ol and 1-dotriacontanol.

Wax extracts from lowermost surface region of the culms were dominated by alkanes of chain length between C₁₈ to C₄₄. The n-alkanes represented 17.5% of total epicuticular waxes from lowermost region of which tritetriacontane was the most copius followed by octacosane. Although, both odd and even number alkanes were present, even number alkanes were most abundant (C₁₈ to C₄₄) and contributed 57.2% and odd number alkanes (C₁₅ to C₄₃) made up 42.8% of the 17.5% of the total alkanes.

Table 1:	Epicuticular wax compound classes of Cyperus pangorei culms and processed culm strands
Degree of freedom: 8, For degree of freedom 8 at 5% level of significance the‘t’ value is 2.30, Values lower than this show statistically significant differences

Alkyl wax esters contributed to major type of constituents of the total epicuticular waxes of the lower most culm regions i.e., about 73.75% of the total waxes. Alkyl esters were of the chain lengths between C₁₃ and C₄₃ and comprised of alkane esters, fatty acid esters and carboxylic acid esters. Fatty acid esters were up to 50% of the total wax esters. The most predominant fatty acid ester was heptadecanoic acid methyl ester (C₁₉). No fatty acids, alcohols and sterols were detected in this region.

The middle region of the culm comprised mostly of very long chain alkyl esters of chain lengths between C₁₅ to C₆₉, followed by alkanes (15%) chain lengths C₁₂ to C₄₃ and fatty alcohols (C₁₅ to C₅₀, 15%); both primary and secondary alcohols were detected. No fatty acids were present and other components like sterols, triterpenoids, alkenes, thiols, ethers and benzopyrones made up the remaining 20%. The most predominating component in middle region of the culm was secondary alcohol; Dodecatrien-1-ol followed by heptacosanoic acid methyl ester and 1-Dotriacontanol. Unlike the lowermost region, wax esters made up only about 50% and were also characterized by the presence of fatty alcohols and sterols. In addition a very long chain wax ester nona-hexacontanoic acid (C₆₉) was detected. The presence of such a very long chain component in plant waxes has not been reported hitherto. Among the alkanes, even chain alkanes were predominant (C₁₂ to C₃₂) similar to that of the lower most region. Alcohols were more abundant in the middle region (15%), all being primary alcohols with only Dodecatrien-1-ol and cyclohexanol being secondary alcohols, the primary alcohols were 1-dotriacontanol, 1-triacontanol and 1-pentacontanol, respectively.

Cuticular waxes of the top region were more or less alike, predominated by alkyl esters (C₁₄ to C₆₉, 51.25%) followed by alkanes (17.5%, C₁₂ to C₄₄), fatty alcohols (2.5%, C₁₆ to C₅₀) and fatty acids (12.5%, C₁₀ to C₂₂) and other trace components like benzopyrones and alkenes as given in Table 1. Fatty acids have been observed only in the uppermost culm region. The most abundant components of total waxes were 1-Dotriacontanol, followed by pentadecanoic acid, heptacosanoic acid methyl ester and tridecanol. The ratio of even chain alkanes was high (71.4%) when compared to odd chain alkanes (28.6%). Fatty acids present include octadecanoic acid, tetradecanoic acid, tridecanoic acid, decanoic acid, eicosanoic acid, hexadecanoic acid, docosanoic acid, pentadecanoic acid and heneicosanoic acid. Primary alcohols were the most abundant of the alcohols and traces of the secondary alcohols such as cyclohexanol were present. No sterols were detected.

In general, the chain lengths remained consistent in all three regions of the culms: alkanes (C₁₂ to C₄₄), wax esters (C₁₄ to C₆₉; C₆₉ absent in the lowermost) and alcohols (C₁₅ to C₅₀) in middle and upper most regions. All three different culm regions were abundant in wax constituents with statistically significant differences in major compound classes (Table 1).

Although epicuticular wax analysis has been extensively studied in dicotyledonous plants (Jeffree, 1996; Dragota and Reiderer, 2007), there are only few studies on monocotyledonous plants such as Hordeum, wheat, sorghum (Tarumoto, 2005; Gorb et al., 2005), Allium, Gloriosa, Yucca and Strelitzia (Barthlott et al., 1998) and none on sedges.

Wax esters are the most abundant components in Cyperus pangorei and have also been reported to be prevalent in other monocotyledonous species such as Musa paradisiaca (Baker et al., 1982), rice, barley (Richardson et al., 2005), maize and sorghum (Gorb et al., 2005). The lowermost region of the culm comprising young tissues is also abundant in waxes and rich in the chemical composition. This illustrates that cuticle formation parallels cell expansion as also in the case of grasses. However, the very long chain fatty acid ester nona- hexacontanoic acid (C₆₉), alcohols and sterols were present only in the mature (middle and upper) regions of the culm, which negates the previous hypothesis that onset of wax deposition occurs at a very early age and developmental stage, however the amount and composition of waxes differ from one plant organ to another with changes during development, season, location and other environmental factors (Jenks et al., 1995; Shepherd and Griffiths, 2006).

Wax morphology: Light microscopic studies revealed that an exceptionally thick cuticle bounded the epidermis measuring c. 100 μm and c. 150 μm, respectively (Fig. 1 and 2).

Fig. 1:	A light microscopic view of the transverse section of the culm of C. pangorei, stained with safranin, showing a very thick cuticle with tooth shaped epidermal cells

Fig. 2:	The epidermal peel of the culm of C. pangorei showing a large amount of stomata with thick wax covering; Stain- Malachite green

SEM examination on culm surface exposed the presence of a thick wax layer (Fig. 3 to 8). Such wax covers the surface as an amorphous film, with irregularly shaped crystals, fissured layers (Fig. 3), granules (4), thick crusts (Fig. 5) and plates (Fig. 6). Abundant wax crystals were found near the stomata both on the dorsal and ventral sides (Fig. 7, 8).

Fig. 3:	An amorphous layer of wax on the surface of the culm with irregular crystals and fissured layers

Fig. 4:	Wax globular structures on the surface of C. pangorei

Fig. 5:	Very thick wax crusts with characteristic morphology

Fig. 6:	Wax platelets with orthogonal symmetry

Fig. 7:	Dense wax deposition and amorphous film on the stomata-on the surface

The epicuticular wax coverage of sedge culm is dense and non-uniform. It consisted of two superimposed layers of waxes, the upper that had crystalline structures and the lower, which was a thick amorphous film. The wax crystals varied greatly in size and form (Fig. 4). No projecting stalks were found. In the lower most region although the crystals were very densely arranged, they remain separated and do not form a particular network (Fig. 3). Dense aggregates and granules were abundant with a characteristic pattern (Fig. 4). These crystals were found attached to a thick amorphous film of wax and also around stomata with wax crystalline structures surrounding the stomatal pore.

Fig. 8:	Dense wax deposition and amorphous film on the stomata- inner side of epidermis

Very thick layers of crusts and fissured layers were not to be seen in the lower most region and the crystals appeared as if they were in the assembly process. The middle and upper region of culms consisted of very compact fissured layers and thick crusts with sculptured appearance (Fig. 5). Very thick waxy plates with orthorhombic symmetry and fissured layers were predominant (Fig. 6).

Assembly of epicuticular waxes exhibit great diversity and are determined by a principle component/dominant class or otherwise the total wax composition (Barthlott et al., 1998; Meusel et al., 2000; Ensikat et al., 2006). The amorphous film of wax assemblage is attributed to the predominance of alkyl esters, alkanes and alcohols. Such films have also been reported from Citrus and Pinus (Jenks et al., 1995). The alkanes form a plain layered structure whereas molecules with terminal polar groups like fatty acids and primary alcohols crystallize in a double layer structure (Koch and Ensikat, 2008). The wax films appear to be ubiquitous while thick layers and crusts are rare. Crusts have been reported from all major groups of plants including Cynanchum sarcostemma, Copernicia cowellii and Buxbaumia viridis (Barthlott et al., 1998; Koch et al., 2009). The formation of crusts over the amorphous film is mainly due to the presence of alkyl wax esters. The abundance of wax esters Cyperus pangorei culms may be contributing to the very thick crusts in the middle and upper regions.

Wax plates with orthorhombic symmetry exhibit one of the most common micro morphology in all type of plants and vary considerably in shape, chemical composition and spatial pattern (Barthlott et al., 2003). The orthorhombic symmetry observed in C. pangorei is similar to that of Yucca platelets (Koch et al., 2006). Even though the wax micro morphologies are implicated by the presence of several types of chemical constituents, the actual chemical composition of any individual crystal is not known.

The wax morphology is also influenced by environmental conditions during the crystallization process and also by the cuticle. Wax deposition is also further influenced by humidity, salinity, cold stress, mechanical stress, altitude and pollution (Shepherd and Griffiths, 2006).

Because of their unique composition, some of the wax esters from natural sources have proven to be important commercial commodities. All plant waxes are harvested more or less directly from plant surfaces, except for jojoba seeds. Plant cuticular waxes are therefore chemically much more diverse than all the other wax sources and this greater chemical diversity goes hand in hand with the variations in wax physical properties that are desirable for industrial applications.

CONCLUSION

The study reveals the rich diversity of waxes in the culms of the sedge Cyperus pangorei. The wax micro morphology demonstrates the fact that the chemical composition influences wax crystal patterns. The enormous amount of waxes as crystals on the surface of the culm may have a role in giving the silky texture to the mats. Further, the abundance and chemical diversity of waxes in Cyperus pangorei may be exploited as an attractive source for important commercial applications.