Sphagnum is one of the important species in the earth; the genus sphagnum has about 100 to 300 species. Sphagnum genus is the major constituent of ombrotrophic bogs contains large amount of living and dead biomass (Van Breemen, 1995, Jim Provan and Pamela J. wilson). Peat lands covers about one third of the global carbon (C) (Gorham 1991). Sphagnum is the major species in peat lands contains large amount of carbon in locked form. Sphagnum is comprise of secondary compounds such as polyphenols and uronic acid, due to this peculiar characters sphagnum is resistant to decomposition of organic matter due to microbes (painter 1991,Verhoevn and Liefveld, 1997, Freeman et al 2001) and the decomposition rate of sphagnum is very less compared to other plants. The carbon is stored in sphagnum in the form of polyphenols. It has a history of usage since from eras to preserve the dead bodies, cheese, etc. due to the peculiar characters of the sphagnum and it is widely used medicinally. They are the secondary metabolites produced in the phenylpropanoid pathway in presence of the enzymes phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase as key enzymes. Phenylpropanoids are the largest secondary metabolites produced by the plants because of biotic or abiotic stresses, UV radiation, temperature and exposure to other environmental conditions (L.G. Korkina, 2007). All species of sphagnum examined are contains high concentrations of the Trans-sphagnum acid, it is synthesized from phenylalanine via the phenylpropanoid pathway (Rasmussen et al 1995). Various genes are responsible for the changes in sphagnum. PAL and C4H genes are major genes responsible for production of phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. FAH1 and F3H are responsible for ferulate-5-hydroxylase and flavanone-3-hydroxylase. Atmospheric nitrogen (N) also has significant effect on the polyphenols, that is increasing levels of nitrogen may be leads to depletion of polyphenols and increases the decomposition rate of sphagnum. However the carbon locked in the sphagnum releases in to the atmosphere may leads to harmful effects and drastically changes the global temperatures. So it is necessary to control this. In order to control the decomposition of sphagnum or to inhibit the synthesis of phenolic compounds by generating the transgenic plants of sphagnum it necessary to know about the polyphenols and their pathways, genes controlling the phenylpropanoid pathway and their expression rates. There are less studies available about the sphagnum so it is necessary take some other plants which already studied, for example Arabidopsis as model species. And also it is important to know the genes coding for the enzyme which is responsible for the synthesis of trans-sphagnum acid by using the methods which are suitable for the gene cloning and analysis.
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The increasing temperatures on the surface of earth is said to be global warming. It is due to the greenhouse gases (water vapor, carbon dioxide (CO2), methane (CH4), ozone (O3) etc.), human activities are major among this, carbon compounds (E.g.: CO2, CH4.) are important greenhouse gases. Decomposition of the plant waste is one of the key sources for the carbon. Peat lands are large pools of carbon. The storage of the carbon depends in relation to production and decomposition (Andy Siegenthaler et al., 2010). The sphagnum genus and its species are the major source for the sequestering carbon and its products. The fallowing graph (figure 1) show that the levels of the carbon dioxide is increasing enormously, and it emphasizing that the increasing concentration of CO2 simultaneously changing the temperature levels.
Figure 1: Global carbon dioxide (CO2) and temperature levels Vs Time.
Carbon storage in peat lands
In the boreal region most of the peat lands are available and it covers about 10% of world vegetative land and contains about 30% of locked carbon (Dixon et al, 1994; Vasander and kettunen 2006) in the world total soil (Post et al., 1982). The accumulated carbons from the last glacial maximum and peat lands are high in the boreal region of the Northern Hemispheres having the atmospheric CO2 is 600ppm (Adams et al., 1990). In the peat mosses genus sphagnum contains large amount of living and dead biomass because Sphagnum is a major wetland plant in the northern peat lands, about one percentage of the earth covered with sphagnum moss or sphagnum peat moss and one third of the global carbon stored in the peat lands. Sphagnum mosses hold the carbon compounds as in locked form. It contains the peculiar polyphenols are derived from the cinnamic acid derivative called trans-sphagnum acid and it produce various secondary metabolites. The compounds mainly synthesized due to the external stresses. The peat lands composed of rich in acidity level and mineral poor conditions (Etherington, 1983), due to this the decomposition of the peat moss is not possible and they are stable for years. Because of this reason the carbon and its compounds still remains in the peat mosses. The sphagnum forms a mat like form which are naturally found in bogs and some of the floating mats blocks the drainage and maintains the bog temperatures (Crum, 1976 ; Niering, 1966) and due to their absorbent nature they also forms the sponge like mass, in spring it act as insulator for ice to prevent melting. As we know the temperatures on the earth are increasing immensely, may be due to human errors or environmental factors and increasing levels it may leads to increases the decomposition of peat mosses. If the locked carbon and its compounds in sphagnum decompose and release into the atmosphere, it leads imbalance in the carbon ecosystem and may possibly doubles the present carbon levels and increases the global temperatures and radiation extremely, which may shows extremely dangerous effects on the all types of organisms living on the earth.
Sphagnum life cycle
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Figure 2: The life cycle process of the genus sphagnum
Sphagnum is the genus in most of the mosses; it belongs to the family Sphagnaceae and a division of bryophyte. The life cycle of sphagnum is quite different from other plant species. The mosses not bear the flowers or seeds. They germinate from the spores and further develop as a fruit like structure called capsules. Sphagnum consists of two different generations of the life cycle they are gametophyte and sporophyte (figure-1). In the gametophyte the plant bears leaves, synthesizes the chlorophyll and it is haploid, it can be able to generate sexually, where sphagnum produce egg and sperm on the branches of the same or different plants. The sporophyte is a diploid generation, which consists of small black capsules on the apex attached to gametophyte and leaves. The spores are enclosed in the capsules and ejects when they are dried. The sporophytes generated from the zygote by sexual reproduction. The haploids produced from the diploid by the process of meiosis. The sporophyte usually spread by spreading the fragments and budding in new plants (figure-2), because most of the species do not reproduce sexually. In some species the fragments persist to dry for long time and still have the nature to grow when water is available, in other species once fragments becomes dry about a week, they cannot grow further (e.g. S.capillifolium) (McQueen 1990). The spores are spread by air. Some plants spread their generation and growth from the branches.
1 2 3
Figure 3: The spreading of the sphagnum from the buds by dispersing the spores and from branches. (1- sphagnum plant bears the buds, 2-spreading the spores from the buds and 3-spreding of the sphagnum from branches)
Sphagnum nature and distribution
Sphagnum mosses are available in two types, sphagnum moss and sphagnum peat moss and they are different. Sphagnum moss is the living form of the sphagnum genus and sphagnum peat moss is the dead mass which accumulates the lower levels of the sphagnum. The dead Sphagnum has the nature of holding the water. It is the major wetland plant, it usually grows on the wet areas or where the moisture content is more. It has a history more than 10,000 year, in olden days the acidic sphagnum bogs are used to preserve the mammalian bodies for ages. These bodies are said to be peat bodies for example Tollund Man, Haraldskaer Women, Lindow Man, etc. Sphagnum plants constitutes of stem, clusters of branches and small leaves, usually it contain both living cells (Chlorophyllose cells) and dead cells (Hyaline cells) with capacity of holding water in large amounts. The sphagnum peat moss can be easily distringwished from the other mosses by its unique branches. Northern Hemisphere is the bulk source for the peat mosses, different kind of species available over there and also present in the South Hemisphere but found less species because of the availability of more lake and humid weather conditions.
Lange and Jensen already reported a survey about the spread of mosses in the greenlands (1887) and the details of sphagna was given by Jensen (1898, 1906, 1910), and Harmsen and Seidenfaden (1932), Harmsen (1933) and Hesselbo (1948) and the authors reports that the availability of various species in different regions, it is mainly reported from the Northern Hemisphere (Bodil Lange). Sphagnum generally finds on the wet areas mainly where the water content is more. It is also observed on the walls where moisture or wetness is more and in the seepages and water pools. Especially where the rainfall is more for example Scotland provides an ideal condition for their growth. It is common in the northern Eurasia and northern North America due to more wet areas. In the Northern Hemisphere the peat moss population available highly and are mainly located I archipelago of Svalbard, Norway of Arctic at 81o N. In the Southern Hemisphere the population of peat moss found in New Zealand, Tasmania, Chile and Argentina.
Taxonomy of sphagnum
Sphagnum genus is present in mosses ranging from 100 to 300. Sphagnum has wide diversity in the natural ecological system. There was number of types of sphagnum species are already listed, but it is still not complete. The genus Sphagnum classified depends on different categories, mainly according to their morphological, chemical, size, color and other characters.
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Table 1: List of few sphagnum species
List of species
These species have different type of characters. They usually have different shapes, sizes of leaves, unique distribution of branches, various types of macro characters and living nature.
Microbial activity of the genus Sphagnum
Sphagnum mosses are remnants to decomposition; it has compounds composed of peculiar chemistry. Sphagnum litter is composed of the specific secondary compounds such as polyphenols and uronic acid; they are resistant to the microbes and protect sphagnum from decomposition. Because of its anti-microbial activity in ancient times it was used to preserve the dead bodies of the humans, animals, etc.
Applications of the sphagnum
Sphagnum has long term history more than 10,000 years of uses for humans, animals and plants because of its natural habitats. Due to its water holding nature sphagnum is used as soil additive and also sale as a soil enhancer, it has potential to improve productivity in sandy soil and to improve the agricultural productivity (Hong Li et al., 2004 and Pulleman et al., 2000). Sphagnum is used as medicinally since 11th century because of its anti-microbial activity (A.P. Podterob and E.V. Zubets). In Europe it has been used in the bandages for the wound dressing and abscesses since. Because of the dry sphagnum has absorbent nature and also used as antiseptic, at the time of first and second world war it was used for wound dressings and also for bedding and babies nappies. The smoke of the sphagnum is used while preparing Scottish whisky and to give favor to it. Currently sphagnum plants are used for the gardening. In Roman times sphagnum was used as a fuel, to cover the top of the houses. Due to its absorbent nature especially it is used as oil absorbent to remove oil spills. In United states its used as oral hygienic product due to its absorbing and bactericidal nature for the tooth paste preparations as a bacterial flora in the oral cavity of mouth (A. P. Podterob and E. V. Zubets, 2002). The species Sphagnum cristatum and Sphagnum subnitens are harvested in New Zealand are exported internationally for the use of hanging basket liners and for orchids as growing supplement. It is also used as bedding material for the animals.
Polyphenols in sphagnum
Polyphenols are the secondary compounds also said to be defensive compounds (Hättenschwiler and Vitousek, 2000). Sphagnum is a rich source for phenolic compounds that act both as structurally and as inhibitors of microbial decomposition of the organic matter (Verhoeven and Liefveld 1997; Freeman et al. 2001). Lignin not present in Sphagnum (Mauseth, 1998), it contain other phenypropanoids as trans-sphagnum acid in bulk (Rasmussen et al. 1995). Trans-sphagnum acid (-p-hydroxyl-fl-[carboxymethyl]-cinnamic acid) is derived from the cinnamic acid unique in peat mosses. Cinnamic acid is the key compound for the synthesis of secondary polyphenols in sphagnum it derived from the phenylalanine. The trans-sphagnum acid is synthesized by phenylpropanoid pathway. The secondary metabolites produced in the phenylpropanoid pathway may include flavonoids, hydroxycinnamic acid ester etc. The analysis of the bog water shows significant amount of trans-sphagnum acid excreted from the sphagnum, it is about 1µM (Wilschke et al., 1989), so it may be necessary to analyze the amount of phenolic compounds persist in the cell wall and their excretion rate. In sphagnum the incorporation of phenolic compounds mask the histochemical identification of cellulose and the in other side the Million's reagent stains the cell wall in to red color. It proves that the sphagnum species do not contain lignin as like higher plants.
Effect of nitrogen on polyphenols
Nitrogen is the chief nutrient, important for the plants and it controls the plant growth. Both PAL1 and PAL2 show the response due to nitrogen and temperature. Studies shows that depletion of the temperature and nitrogen levels leads to two-fold increase in PAL1 and PAL2 transcripts. It makes a prediction that increasing levels of nitrogen in the sphagnum leads to decreasing the concentration of polyphenols. The sphagnum bogs get their nutrition from the atmosphere; it creates an hypothesis that increasing levels of the nitrogen in the atmosphere may alter the chemistry of the litter and carbon balance globally. Nitrogen is responsible for the amino acids and nucleotide formation (Sittt and Krapp, 1999). The alterations in the carbon and nitrogen metabolism may change the phenylpropanoid pathway. In the addition of nitrogen to the nitrogen-depleted plants shows alteration in the expression of hundreds of gens (Scheible et al., 1997a, 2000; Scheible et al., 2004; stitt, 1999; Wang et al., 2000, 2003, 2004). Polyphenols has strong inhibitory effect against the microbes, the increasing availability of atmospheric nitrogen favors for microbial metabolism and accelerate the decomposition of peat moss or litter. But, it leads to the emission of higher amount of carbon dioxide (CO2) and other carbon related products subsequently causes global warming.
Pathway of polyphenol synthesis in sphagnum (Phenylpropanoid pathway)
Phenolic compounds are unique in all peat mosses but the concentration of trans-sphagnum acid varies from the species (Rudolph and Samland., 1985). Phenylpropanoids are organic compounds synthesized from cinnamic acid obtained from the phenylalanine in presence of the enzyme phenylalanine ammonia-lyase (PAL). Phenypropanoid pathway is unique in all vascular plants (Lewis and Yamamoto, 1990). Wide ranges of metabolites are produced in this pathway and they are contributing their effect in plant defense mechanism (Nicholson and Hammerschmidt, 1992) and other environmental stresses. The compound include lignins, flavonoids, isoflavonoids and phytoalexins. 4CL (4-coumarate: CoA ligase) is an ester derived from cinnamate (Klaus Hahlbrock and Dierk Scheel., 1989). Cinnamic acid is the key compound for the synthesis of secondary metabolites in all the plants. The polyphenols produced after a series of reactions as hydroxylation, methylation and dehydration and produces pCumaric acid, caffeic acid, ferulic and sinapic acid and simple coumarins. Phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase are the key enzymes in the phenylpropanoid pathway to produce secondary metabolites.
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Figure 4: The general pathway of polyphenols in plants (Phenylpropanoid pathyay)
Phenylalanine ammonia lyase (PAL)
It is usually present in all the green plants and it is the first enzyme involved in the phenylpropanoid pathway. It's an important enzyme and most studied enzyme for the secondary metabolism in phenylpropanoid pathway. It is the precursor for the major constituents, it converts phenylalanine in to trans-cinnamic acid in sphagnum.
Cinnamic acid 4-hydroxylase
Cinnamate 4-hydroxylase belongs to cytochrome P450 monooxygenase, it invalves in the first step of oxidation in the phenylpropanoid pathway, it catalyzes the oxidation step and plays key role in the synthesis variety of secondary metabolites. It is the second enzyme involved in the phenylpropanoid pathway. It converts trans-sphagnum acid in totrans-p-coumaric acid and also controls the fluctuation of the carbon and protects the plant from pathogens and UV radiation. C4H mainly produces in response to light and plant defense state like wounding and infections. The compounds formed due to C4H mechanism are essential for lignification and defense mechanism
Genes controlling the Phenylpropanoid pathway in sphagnum
Various genes controlling the enzymes involved in the phenypropanoid pathway. It is possible to analyze the genes by purifying the enzymes and cloning the respective genes. These genes already isolated fom various plant organisms. The enzymes involved in the phenypropsnoid pathway of sphagnum are phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, trans-sphagnum acid synthase. Except the enzyme trans-sphagnum acid synthase the other enzymes are already studied in different plants for example Arabidopsis. PAL and C4H genes are important and they code the key enzymes phenyalanine ammonia lyase and cinnamic acid 4-hydroxylase. The FAH1 and F3H genes are responsible for the synthesis of flavonoids, they codes for the ferulate-5-hydroxylase and flavanone-3-hydroxylase. The PAL, C4H and flavonoid genes are produce in response to biotic or abiotic stresses, temperatures and external environmental factors. The genes PAL1 and PAL2 produce response to nitrogen and temperature, depletion of these environmental factors increases the PAL1 and PAL2 transcripts. To understand much about the role of PAL gens in the regulation of phenylpropanoid pathway the PAL gene from French been (Phaseolus vulgaris) introduced into the tobacco and studied the consequences of the enzyme and its overexpression (Elkind et al., 1990). It shows reduced endogenous PAL genes expression in both RNA and enzymatic activity its due to sense suppression. The two genes C4H and ferulate-5-hydroxylase are difficult to aim due to the protein unstability. The gene coding for the C4H was recognized (Fahrendorf and Dixon, 1993; Mizutani et al., 1993; Teutsch et al., 1993) and purified the enzyme (Gabriac et al., 1991; Mizutani et al., 1993) and the gene encoding for ferulate-5-hydroxylase was cloned by tagging T-DNA (Meyer et al., 1996b).
The cDNA of the C4H gene was isolated using reverse transcriptase-PCR and RACE-PCR (Rapid amplification of the cDNA ends PCR) from Korean native bramble (Rubus coreanus Mique). In Arabidopsis cDNA isolated for a cytochrome P450, cinnamate 4-hydroxylase using C4H cDNA of mung bean as a probe. However the performance of C4H expression with other gens involved in phenylpropanoid pathway is still unknown. The sequence analysis of 5' promoter region of the C4H gene clone realizes that the expression of PAL and 4CL genes is regulated due to the cis-elements involved in this process (Longmann et al., 1995) also be present in the C4H promoter.
Over expression of PAL and C4H genes
The overexpression of PAL genes reported in different plants. The enzyme L-phenylalanine ammonia-lyae was overexpressed in the transgenic tobacco contaiing PAL2 genes of been shows silencing of the endogenious PAL genes of tobacco (Paul A. Howles et al., 1996). In the trans genic roots of C.blumei containing the Arabidopsis thaliana PAL1 gene, and controlling under CaMV 35S promoter shows the lowering the activity of phenylalanine ammonia lyase and also decreses the amount of phenolics, rosmarinic acid and chlorogenic acid. The F5H overexpression in Arabidopsis under control of cinnamic acid 4-hydroxylase as apromoter shows significant changes in F5H expression (Knut Meyer et al., 1997). In Arabidopsis the C4H expression was analyzed using RNA blot hybridization, it shows modest increase in C4H expression (Dolly A. Bell-Lelong et al., 1997). The above evidences creating an hypothesis that the over expression of PAL and C4H genes in sphagnum may alter the phenylpropanoid pathway and perhaps decrease the production of the phenolic compounds
Arabidopsis as a model species for sphagnum
Arabidopsis is a model plant for number of researches, it is a small and flowering plant and contains smallest gene. Because of its wide range of studies it is known as model organism for various types of gene related studies. To understand the various gene related issues in sphagnum, Arabidopsis is taken as a model, because it is the organism of choice. The plan Arabidopsis provides a global gene expression data because it exposed to various treatments include light, temperature and salt or dehydration. The meta-analysis of the Arabidopsis provides potential offers to determine cellular process and its associated genes and also unknown genes. The phenylpropanoid pathway in Arabidopsis contains three fluorescent sinapate esters (sinapoylglucose, sinapoylmalate and sinapoylcholine). In arabidopsi the phenyalanine ammonia-lyase is coded by four genes. They are PAL1, PAL2, PAL3 and PAL4. The PAL1 and PAL2 contains almost identical coding region, but in PAL3 and PAL4 it shows less similarity (E Logemann et al.,). The quantitative real time PCR studies shows the changes in C$H gene patterns (Baek MH et al., 2008).
Brief experimental protocol
Sphagnum is an important plant in the natural ecosystem and it is necessary to understand the genetic construction and changes to investigate polyphenols in it. In order to understand about the genetic control of the phenotype, it is necessary to predict or hypothesis can be tested, so in the fallowing experiment propose to investigation by the hypothesis (based on the genetic construct).
From the above details PAL and C4H genes are important in phenylpropanoid pathway, these genes already cloned and sequenced from Arabidopsis thaliana. In the experiment we will use the coding sequence of these to isolate homologues from sphagnum. We initially fallow the known Arabidopsis genes to search for corresponding homologues sphagnum using southern blot. It will allow us to estimate the number of sphagnum genes hybridizing to the Arabidopsis gene sequences, their copy and approximate size of them. Then we will use the degenerate primers constructed for Arabidopsis PAL and C4H genes and PCR to amplify the corresponding sphagnum homologues, then which will cloned into the plasmid or phage vectors. These cloned genes will be sequenced, and used in northern hybridizations on sphagnum to assay for the expression profile of these genes. And at this stage we will have isolated sphagnum genes involved in the synthesis of trans-sphagnum acid and determine their expression profile. In this experiment we will use a new cloning technique that is The TOPO® Cloning technology
The TOPO® Cloning technology
The enzyme DNA topoisomerase I is the key key enzyme in this technology, it works as a restriction enzyme and as a ligase. The principle involved that the pentameric sequences 5'-(C/T)CCTT-3' is recognized especially by Vaccinia virous topoisomerase I and forms the covalent bonds with phosphate group attached to the 3' thymidine and cleaves one DNA strand leads to unwind the DNA. To bind the transfer activity of topoisomerase, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3´ phosphate. This enables the vectors to readily ligate DNA sequences with compatible ends. The ligation is complete in only 5 minutes at room temperature (Suzanne M. Paquette et al., May 2000).
The key objectives of this experiment
To stabilize the concentration of the greenhouse gas in the atmosphere by inhibiting the release of locked carbon from the sphagnum.
Bodil Lange. The genus sphagnum in Greenland, June-1952, the Bryologist. Vol.55. No.2.
A.P. Podterob and E. V. Zubets. A History of Medicinal Use of Plants of Genus Sphagnum. Medical Plants, Pharmaceutical Chemistry Journal, Vol. 36, No. 4, 2002.
Suzanne M. Paquette, Søren Bak, René Feyereisen. DNA and Cell Biology. May 2000, 19(5): 307-317.