Accelerate Peat Land Photosynthetic Carbon Acquisition Biology Essay

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This is a Literature Review Plan Submitted to the Faculty of Biological School in Partial Fulfilment of the Requirements for the Degree of Master of Science in Molecular Biology with Biotechnology at Bangor University.

1 Introduction

2 Review of the literature

2.1 Global warming

The phrase 'Global warming' represents a wider meaning which entails the anthropogenic effect on the climate, by the burning of fossil fuels and large-scale deforestation, which releases cause large amounts of 'greenhouse gases', such as carbon dioxide . The rise in the temperature and its projected continuation due to the increase in atmospheric concentration of carbon dioxide emissions is predicted to lead to significant changes in climate (Cox et al. 2000).

As a result, one such component of global warming is carbon dioxide (CO2) emissions, the most important of greenhouse gases, influenced directly by anthropogenic activities. Gases such as CO2 absorb infrared radiation emitted by the Earth's surface and act as blankets over the surface keeping it warmer than it would otherwise be (Cox et al. 2000).

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The increase in atmospheric carbon dioxide is well documented by ecologists and studies, and reports show that carbon dioxide levels have been increasing at an alarming rate. According to Intergovernmental Panel on Climate Change Assessment Report (2001) (IPCC 2001), the earth's mean anticipated temperature increase in the twenty-first century is between 1.5 - 5.8 °C. The leading and direct effect of the rise in atmospheric carbon dioxide will be increased decomposition rates of organic matter which will in turn release CO2 to the atmosphere leading to an enhanced global warming trend (Figure 1). This is a major cause for concern because such feedback loop can me important in determining quantitative amount of discharge in the atmosphere, because as such, it is known that CO2 is responsible for 55% of increase in rising temperatures due to anthropogenic activities and also there is twice as much carbon stored in the soil as in the atmosphere (Cox et al. 2000, Freeman et al. 2004a). The accelerated decomposition of organic would undoubtedly add to the already ever increasing rise of the CO2 levels in the atmosphere.

In a study by Freeman et al (2004a) discovered that compared with the control cases, there was a ten times increase in the proportion of dissolved organic carbon derived from recently assimilated CO2 when CO2 levels were increased. They found that the environmental factors have a stronger impact on the concentrations of dissolved organic carbon on the effect of primary productivity compared effects on decomposition alone. Human activities have significantly altered the nitrogen and carbon cycle. In the light of the current concerns on the environmental changes and the modifications in the nutrient cycle having major implications on the natural ecosystem has lead to the interest in identifying existing and potential carbon sinks (Euliss et al. 2006).

Figure 1 Effect of global warming on changes in land carbon storage. The red lines represent the fully coupled climate/carbon-cycle simulation, and the blue lines are from the `offline' simulation which neglects direct CO2-induced climate change. The figure shows simulated changes in vegetation carbon (a) and soil carbon (b) for the global land area (continuous lines) and South America alone (dashed lines).

Figure 1 Monthly mean carbon dioxide globally averaged over marine surface sites. The dashed red line represents the monthly mean values, centered on the middle of each month. The black line represents the same, after correction for the average seasonal cycle. The latter is determined as a moving average of SEVEN adjacent seasonal cycles centered on the month to be corrected, except for the first and last THREE and one-half years of the record, where the seasonal cycle has been averaged over the first and last SEVEN years, respectively. [Adapted from http://www.esrl.noaa.gov/gmd/ccgg/trends/global.html#global

2.2 Carbon storage in wetlands

The Ramsar Convention (1971) defines wetlands as:

"...areas of marsh, fen, peatland or water, whether natural or artificial, permanent or temporary, with water that is static or flowing, fresh, brackish or salt, including areas of marine water the depth of which at low tide does not exceed six metres".

They include marshes, swamps, peatlands (including bogs and fens), flood meadows, lakes and ponds, rivers and streams, estuaries and other coastal waters. All wetlands are considered important marine ecosystems for carbon sequestration. Although wetlands occupy a small portion of the earth's surface, a large portion of the carbon is stored in the`1 soil reservoir (~1500Ã-1015 gC) (Schlesinger 1991). The accumulation of carbon in wetlands is determined by the difference in the primary production and the decomposition of organic material and carbon is stored in wetland sediments over the long term. Short-term stores are in existing biomass (plants, animals, bacteria and fungi) and dissolved components in the surface and groundwater.

2.2.1 Peatlands

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Peatlands are characterized by waterlogged conditions and dominated by hydrophytes. Peat consists of the remains of animals and plant constituents that accumulate in water saturated conditions as a result of incomplete decomposition. The factors that cause peat to accumulate vary depending on soil, climate and plant species. Usually on relatively flat landscapes, peat accumulates to depths of more than 30 or 40 cm and often up to several metres (Gorham 1991).

The waterlogged and anoxic conditions a few centimetres beneath the surface prevent organic detritus from rapidly decomposing and results in net accumulation of carbon making peatlands important stockpiles of locked carbon. It is estimated that 3% of the earth's land area is covered by peatlands of which 350-535 Gt of carbon, or between 20 to 25% of the world's soil organic carbon stock (Gorham 1991) is locked up and most of the peatlands are found in the Northern Hemisphere.

In wet climates, the peat surface derives nutrients solely from the atmosphere as the peatlands are arched above the surrounding landscape. In this case, the peat consist the bulk of the remains of Sphagnum mosses referred to as ombrotrophic bogs (Gorham 1991) and are characterized by a dense cover of Sphagnum mosses, which is well adapted to such environmental conditions. Ombrotrophic bogs are sinks of carbon store and supply 455 gigatonnes of carbon globally, amounting to between 20 to 30% of all the soil carbon on earth (Gorham 1991) and are. In the UK, around 8% is covered with upland peatland, combined with Ireland, this amounts to 15% of the total world peat bog.

The peatlands are one ecosystem what will be affected by the recent changes in climate and nutrient alteration through changes in the rate of organic carbon (peat) storage.

2.3 Sphagnum as a major wetland plant

2.3.1 Sphagnum taxonomy, morphology, and ecology

Sphagnum falls under the phylum Bryophyta which consists of mosses, liverworts and hornworts (Shaw et al. 2010, Margulis, Chapman 1998). Mosses are non-flowering plants and reproduce via spores. The genus Sphagnum is divided by following the factors: cell structure; the number of branches in a fascicle; branch orientation, shape and colour; and habitat (McQueen 1990, Flatberg 2002).

The genus Sphagnum (peat mosses) includes about 350 - 500 recognised species but only about fifty are important in peat formation (Shaw et al. 2010, Gunnarsson, Shaw & Lonn 2007). A comprehensive cataloguing of the mosses in Europe was done by (Hill et al. 2003). Following Hill, 51 Sphagnum species are currently recognized in Europe, most of which occur in north Western Europe while in North America, 84 species were identified by (Halsey, Vitt & Gignac 2000). In European bogs, the common colonising Sphagnum species are S. magellanicum and S. papillosum. (Fig.1). The hummock species S. fuscum, S. rubellum and S. russowii and hollow species S. balticum and S. magellanicum are characteristics of ombrotrophic (rain-fed) bogs in Scandinavia (Malmer 1988, Money, Wheeler 1999). Pigmentation can vary in the Sphagnum species but most of them are green but pigmentation can appear also as yellow, brown, orange or red (Daniels, Eddy 1985) .

Mature spores of Sphagnum germinate in to plants (Fig.2a) through a stem elongation. The main stem, 5 - 10cm long, is surrounded by one to five layers of hyaline cells with various reinforcing cell wall fibrils and pores and a central region and the arrangement of the leaves is interspaced with smaller photosynthetic cells. They are dead, hollow, (a) with spiral thickenings and circular pores (c) and interspaced with smaller, elongated cells that contain chloroplasts (b) and surround the hyaline cells; located in the plant leaves (Fig.2b).

The hyaline cells are morphologically adapted to retain large quantities of water through soil and the atmosphere (McQueen 1990) and vary between species. In other species, the dead cells of the stem and the leaves hold water by capillary action, while in others the twisting of branches around the main stem forms a structure that functions as a wick. This trait is essential for water uptake through soil moisture as these plant species lack roots or internal water carrying tissues for water transport. Due to these traits, Sphagnum moss plants are very efficient in retaining water and can store hold up to 20 times their dry weight of water, it is this feature that causes them to be sponge like (Clymo, Hayward 1982). The Sphagnum moss species are divided into three categories depending on water content: hummock if the (water level deeper than 20 centimetres), intermediate (water level between5-20 cm) and flark-level species (less than 5 cm) (Daniels, Eddy 1985) .

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The morphology of each species depends on a number of factors, most notably wetness and exposure to habitat (light and pH). Sphagnum plants grow into masses that are closely packed communities and form floating mats that often form hummocks with hollows in between. The mats are composed of several leafy stems that grow vertically with a shoot density 1 - 7 shoots cm-2 depending on species however in permanent waterlogged conditions, they grow horizontally (Rydin, Jeglum 2006). Different Sphagnum species dominate hummocks while others prefer wetter hollows. This floating keeps the bogs cool, in spring it serves as an insulator while in summer it absorbs water from the surrounding. There is a cluster of branches called capitulum at the top of each branch with has more side branches than on the lower stem (Fig.?). It is in the cell walls of the capitulum where the reddish-violet pigments synthesised by the Sphagnum plant are deposited.

The growth of Sphagnum depends on the availability of nutrients and water supply. When the lower portion of the Sphagnum plant becomes older, it falls downwards, and these remains can form the peat deposits which can be up to 6cm in height (Clymo, Hayward 1982, Clymo, Hayward 1982, Rydin, Jeglum 2006).

Figure 1 S. magellanicum (From H J B Birks in Handbook of European Sphagna)

Figure 2a Sphagnum spp. The overview (from McQueen 1990).

Figure 2b Typical leaf cell arrangement, hyaline and

chloroplasts

2.3.2 Sphagnum moss chemistry

An additional factor influencing the distribution of Sphagnum species is its chemistry, the main source of water and chemical elements in ombrothophic bogs is solely from atmospheric deposition and are characterized by acidic and nutrient-poor waters. The acidic characteristic is important in distinguishing Sphagnum-dominated peatlands from other peat-accumulating wetlands.

As mentioned earlier, Sphagnum exchanges hydrogen ions for nutrients such as K+, Na+, Ca2+, Mg2+ and thus acidifying the surrounding area. Vascular plants cannot tolerate the acidic and nutrient poor environment so there is no competition for nutrients for Sphagnum. In Sphagnum rich-bogs, the nutrient chemistry is characterised by the availability of nitrogen (N), potassium (K), magnesium (Mg) and phosphorus (P). However, only Nitrogen (N) and phosphorus (P) are the growth- limiting nutrients (Vitt et al. 2003, Turetsky 2003, Bragazza, Freeman 2007b) and the main source of nitrogen for Sphagnum ecosystems are atmospheric deposition, biological fixation, and weathering (Vitt et al. 2003, Turetsky 2003).

Continuously growing Sphagnum plants create sites for ion exchange and the major constituent at those ion exchange sites are high concentrations of uronic acid, which is a cation exchanger (Clymo, Hayward 1982). The uronic acid is held in the cell walls as a polymer sphagnan which make up 10 - 30 % of the dry mass in Sphagnum.

2.3.3 Carbon storage in Sphagnum

As mentioned earlier, Sphagnum is the dominant moss genus found in northern peatlands in which large amounts of carbon sequestered from the atmosphere is stored and not yet released by decomposition (Euliss et al. 2006). It represents about 10 - 15% of the terrestrial carbon locked up as dead and living Sphagnum (Clymo, Hayward 1982). The waterlogged soil forms an anoxic condition favours carbon sequestration, where growth of microorganisms is depressed allowing for the slow decomposition process. In exchange for hydrogen ions, Sphagnum receives its nutrients from the surrounding environment; this increases the acidic condition, as low as pH 3, of the surrounding water (McQueen 1990). This condition fosters the growth of the dead remains of the Sphagnum mosses which pile up and compress together to form peat soil.

It is probably the only bryophyte known to accumulate more carbon than any other plant genus, therefore, the factors effecting the accumulation of Sphagnum peat are of significant importance in view of the change in the global carbon cycle (Clymo, Hayward 1982). A concern highlighted by Gorman (1991) was that the decomposition process may increase in response to elevated global temperature changes such that rather than sinks these wetlands could become sources of carbon dioxide release or the effect of sinks will lessen.

Sphagnum moss has been referred to as a unique ecosystem engineer responsible for the development of peatland ecosystem (??). Consequently, it is an important species to study because of its wide distribution of Sphagnum moss in the Northern hemisphere and its role in the carbon stock. Therefore, carbon sequestration in peatlands strongly depends on Sphagnum mass growth some figures???

2.4 Decomposition of Sphagnum and release of stored carbon

2.4.1 The phenomenon of slow decomposition of Sphagnum litter

As mentioned earlier, Sphagnum typically dominate peat lands fed by atmospheric deposition, and are termed ombrotrophic bogs (Euliss et al. 2006). A significant feature of the Sphagnum moss is its slow decomposition rate through water retention and acidification. It produces phenol rich material that provide structural support and is more resistant to microbial decomposition than the other litter of bog plants (Verhoeven, Toth 1995, Freeman et al. 2004b). In addition, it produces another important polypheonol which is genus specific, p-hydroxy-b-carboxymethyl-cinnamic-acid which inhibits the microbial breakdown hence slowing down the decay of litter of both Sphagnum and other plants (Rasmussen, Peters & Rudolph 1995b, Rasmussen, Wolff & Rudolph 1996, Rudolph, Samland 1985). Thus by creating this acid condition and anoxic condition, Sphagnum strongly reduces microbial degradation of the litter.

It is hypothesized that (i) raised CO2 concentrations cause increased Sphagnum growth, but that (ii) higher N inputs would have an opposite effect by stimulating the growth of vascular plants.

In general, the decomposition rate of Sphagnum litter is 10-20 %ïƒ- per year compared to 40-80 %

per year for vascular plants (Kulzer et al. 2001). In the Polar Regions, Sphagnum moss is decomposed much slower, about 3-5 % per year or even less (Table 2.2)

Table 1 Decomposition rates of Polar mosses. (Adapted from Bowden et al. 1999 (Group 1999)

Rate (% year-1)

Species

Location

Reference

0.04 - 3

Moss, different spp.

Arctic

Russell 1990

5

Sphagnum fuscum

Arctic

Roswall et.al 1975

4

Sphagnum balticum

7

Sphagnum lindbergii

5

Drepanocladus schulzii

7

Dicranum elongatum

1.5

Moss, different spp.

Antartica

Davis 1986

1.3 -2.4

Baker 1972

0.10 -8.3

Fenton 1980

2

Chorisodontium aciphyllum

Signy Island

Collins 1973

14

Sanionia uncinata (dry)

(South Orkney Islands, Antartic)

25

Sphagnum uncinata (wet)

2.4.2 Nitrogen concentration

Decomposition rates are affected by nutrient levels in the plants and changes in the ratios can have a considerable effect on carbon balance. Although the increased CO2 levels do not have any significant effect on the overall effect, but analysis revealed that the CO2 increase significantly reduced Nitrogen concentration in Sphagnum which reduced the polyphenols. Further, Limpens & Berendse (2003) report that in Sphagnum the N:C is ratio is important and effects litter quality, with an increasing decomposition when the N:C ratio is higher which is supported by results derived from {{11 Berendse,Frank 2001;}} where they found that increased N inputs had a significant overall effect on Sphagnum productivity as polyphenols and protein biosynthesis both compete for the same precursor.

Furthermore a number of research studies (Aerts, Wallen & Malmer 1992, Limpens, Berendse 2003, Bragazza, Freeman 2007a) have als shown that increasing nitrogen concentration in Sphagnum litter (as consequence of increased exogenous nitrogen availability) is accompanied by a decreasing concentration of polyphenols. Bragazza & Freeman (2007a) state in their paper that peat decomposition would accelerate if the content of polyphenols is lowered thereby releasing the stored carbon.

Sphagnum, as an ecosystem engineer, seems quite important in the decomposition process.

2.5 Polyphenols in Sphagnum

Plants produce great variety organic compounds called secondary metabolites, one of which is a class of aromatic compounds called polyphenols. These include phenols, phenolic acids, flavonoids, lignin, tannins and others and are involved in plant development, growth and defence. However, the structure of these phenols in Sphagnum species are distinctly different as compared to other vascular plants because of high content of p-hydroxyphenyl and carbonyl groups (Abbott et al. 2010). The group further report four phenols, methylated 4-isopropenylphenol, ethylated cis and trans 3-(4'-hydroxyphen-1-yl)-but-2-enoic acid and methylated 3-(4'-hydroxyphen- 1-yl)-but-3-enoic acid found to be exclusive to species of the Sphagnum mosses.

These hydroxyl phenyl units build a protect Sphagnum cellulose against breakdown by microorganisms. The water soluble carbonyl group exist as phenolic acid specially Sphagnum acid (Verhoeven, Toth 1995, Verhoeven, Liefveld 1997). The excreted water soluble phenolic acids accumulate in bog waters as humic acid which prevents assimilation of the Sphagnum plant material. This acidity prevents other plant form from growing in the surrounding waters. Additionally, Sphagnum acid has identified as a preservative and the breakdown products of the acid discovered in peat bog bodies example the Lindowman {{22 Painter,Terence J. 1991;}}.

Sphagnum contains a diversity of phenols with different molecular weight, most of which have not been fully characterized yet. Higher molecular weight phenolics are present in peat bogs in hydrolysable and solid forms. Although Sphagnum does not contain lignin, the phenolic trans-sphagnum acid is synthesised via the phenylpropanoid pathway from phenylalanine (Rasmussen, Wolff & Rudolph 1996, Rasmussen, Wolff & Rudolph 1995). Correspondingly, phenolics possess important antimicrobial properties. The anitimicrobial properties of the phenolic compounds in various species of Sphagnum (Basile et al. 1999, Mellegård et al. 2009) and in the Indian native moss Sphagnum junghuhnianum (Singh et al. 2007) have been studied. Phenolics are accumulated in bog litter because they are made unavailable to microorganisms. As a result the high molecular weight phenols form a tight link with organic matter thus slowing down decomposition.

2.6 Pathway of polyphenol synthesis in Sphagnum

2.6.1 General Phenylpropanoid biosynthesis pathway

A large class of plant phenols are the Phenylpropanoids, which are produced through the shikimic acid pathway. The phenylpropanoid pathways (Fig. ?) and the functional diversity of their products has long been the centre of attention in plant physiology as it gives rise to a wide variety of metabolites in plant cells. The phenylpropanoid pathway is responsible for the synthesis of naturally occurring polyphenolic coumpounds such as aurones, flavonols, flavones, catechins, anthocyanins, isoflavonoids, dihydroflavonols, proanthocyaaidin (tannins) (JunLi, Jie & GuangYuan 2009) and are involved in many aspects of plant development, pigment production, UV light protection and disease resistance.

There are several genes involved in the phenylpropanoid biosynthesis pathway and several studies (Hahlbrock, Scheel 1989, Dixon, Paiva 1995, Cochrane, Davin & Lewis 2004, Kervinen et al. 1997) regarding the isolation, cloning and characterization of the genes involved in the phenylpropanoid pathway in plants have been carried out. Though it has been possible to clone the genes involved in the pathway and much research has been conducted in cloning these genes from several plant species, the pattern and extent of accumulation of the end products from the pathway determined by the individual enzymes encoded by these genes is yet to be determined. Thus far, only correlations have been used to describe the pattern of accumulation through changes in activity and product level (Hahlbrock, Scheel 1989).

Although the pp pathway involves a number of enzymes to generate the final product of metabolism, the central/primary reactions of phenylpropanoid metabolism involve three enzymes, However, the common enzymes involved in the initial stage of the general pathway are the Phenylalanine ammonia lyase (PAL), Cinnamate 4-Hydroxylase (C4H) and coumarate CoA ligase (4CL) of which the first two will be discussed further in detail.

PAL is involved in the catalysis of the deamination of phenylalanine to produce the product cinnamate, which is converted to 4-coumarate by an oxidative reaction of C4H. The 4 coumarate is then converted to p-coumaroyl-CoA by 4CL which then moves into the branched pathway giving rise to a number of products as mentioned above. Studies have focused on these enzymes, given the importance of both PAL and C4H as key enzymes in the biosynthesis of flavoids, studies have focused on the enzymes as models to understand the regulation of the flux control in the pathway and also for biotechnological manipulations of product accumulation

As mentioned earlier, all sphagnum species are characterized by a large amount of free endogenous trans sphagnum acid (Rasmussen, Peters & Rudolph 1995b) (-p-hydroxyl-fl-[carboxmethyl]-cinnamic acid), a cinnamic acid derivative unique to peat mosses. It is well documented (Rasmussen, Wolff & Rudolph 1996, Tutschek 1982) that sphagnum acid is derived from the phenylpropanoid pathway from phenylalanine. They are excreted in surrounding media as metabolites in the process of Sphagnum growth (Rasmussen, Peters & Rudolph 1995a) suggests the following synthetic route:

Phenylalanine > (Phenylalanine ammonia lyase) > Trans-cinnamic acid > (Cinnamate 4 hydroxylase) > pCoumaric acid > (Tran-sphagnum acid synthase) > Trans-sphagnum acid.

The biosynthesis of and degradation of the most important phenolics of Sphagnum is presented in Fig.?? (Rasmussen, Peters & Rudolph 1995a).

Although the enzyme trans-sphagnum acid synthase remains to be isolated, the genes coding for the other enzymes such as PAL and C4H have already have been isolated from a variety of plant species, including Arabidopsis (Winkel-Shirley 1999),PAL and C4H.

General Phenylpropanoid

Pathway

Flavonoid Pathway

Figure ???? Schematic diagram of part of the phenylpropanoid biosynthesis pathway. Relative enzymes are indicated alongside the appropriate part of the pathway. Continuous arrow, single step; dashed arrow, multiple steps. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; HQT, hydrocycinnamoyl-CoA quintae transferase; GT/ RT, glucosyl transferase/rhamnosyl transferase. (JunLi, Jie & GuangYuan 2009)

2.6.2 Phenylalanine ammonia lyase (PAL) in Arabidopsis thaliana

The Phenylalanine ammonia lyase (PAL; E.C. 4.3.1.5) enzyme has been the subject of much interest (Hahlbrock, Scheel 1989) since its discovery in 1961 (Koukol, Conn 1961). It is known that in higher plants, PAL has functions in plant growth regulation, disease resistance (Hahlbrock, Scheel 1989) and is required for suberin and lignin biosynthesis (Tutschek 1982). It is a key enzyme that links the primary and secondary metabolism and catalyses the deamination of L-phenylalanine to cinnamic acid in the first step of the phenylpropanoid pathway.

. Several factors are known to affect the expression and activity of PAL. They are light, wounding (31), disease, gamma-ray irradiation, germination, development and differentiation, and the application of certain macromolecules (37). Many of plant-derived phenolic compounds (flavonoids, isoflavonoids, coumarines, and lignans) are secondary products of PPs metabolism (17, 18).

The enzyme phenylalanine ammonia-lyase (PAL) is found to be encoded by a small multigene family. In barley, five different PAL genes have been found (Kervinen et al. 1997), two in parsley (Logemann, Parniske & Hahlbrock 1995) and rice (Zhu et al. 1995), Arabidopsis thaliana (L.) Heynh has four putative PAL isoenzymes (Cochrane, Davin & Lewis 2004, Kervinen et al. 1997) with the exception of potato, containing 40 PAL genes (?).

This family of enzymes comprises of many isoforms and are responsible for different branches of the phenylpropanoid pathway and responsive to different development and environmental stimuli. Furthermore, the levels of PAL mRNA and enzyme levels may vary considerably in plant tissues because they are highly regulated during development as well as in response to a wide variety of stimuli and the transcripts of individual. This suggests that species possessing multiple forms of the PAL isoforms regulate the fluctuation into various branches of the phenylpropanoid pathway.

PAL gene show highly different patterns of accumulation implying that that there is a tight control on the initiation and suppression of transcription PAL genes. This suggests that the control of the flux of metabolites through the different branches of the phenylpropanoid pathway is depended on the association of the different forms of the PAL genes with the enzymes complexes (Ohl et al. 1990). It is known that the activity and expression of is affected inresponse to biotic and abiotic stress such as pathogen attack, germination, UV light and plant development and UV radiation (Tutschek 1982). Studies have led to a better understanding of the role of this enzyme in the regulation of the phenylpropanoid metabolism by examining the over expression of a PAL gene by introducing into plants such as tobacco (JunLi, Jie & GuangYuan 2009, Howles et al. 1996, Franke et al. 2000, Chang, Luo & He 2009)

The biochemical characterization of PAL has been extensive reviewed for plant species such as Arabidopsis (Ohl et al. 1990). Of the four PAL genes of A. thaliana, three have been cloned and sequenced (Wanner et al. 1995). It was thought that the first A. Thaliana gene PAL1 was involved in lignifications (Ohl et al. 1990) but found that EST (Costa et al., 2003) data does not support this and that more likely PAL2 is the actual candidate or that both PAL1 and PAL2 are involved. Longemann et al. (1995) state that three sequence motifs (boxes P, A, and L) in the promoter regions of PAL1 genes for responding to UV irradiation have also been discovered in most known PAL gene promoters. Cloning of PAL1 and PAL2 genes from A. thaliana revealed that it is related to PAL from other species and that the promoter region is highly conserved with putative regulatory elements; a single intron and a long highly conserved second exon (Ohl et al. 1990, Wanner et al. 1995).

The other A. thaliana PAL gene clones were identified and obtained from a 2EMBL4 genomic library in Columbia using a parsley PAL cDNA as probe (Wanner et al. 1995). Figure 1 shows comparative maps of all three PAL genes. Following Wanner et al (1995), it was found that there is a notable difference between the first two genes and PAL3 with other sequenced plant PAL genes with PAL3 containing an additional intron and its amino acid sequence less homologous as compared to the other PAL proteins and lacking also the conserved sequence motifs as in the A. thaliana PAL1 and PAL2. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism.

Figure??? Maps of the three A. thaliana PAL genes. The sequenced regions of snbclones coveringA, thaliana PAL1, PAL2

and PAL3 are depicted, including the 5'-upstream promoter region, complete amino acid coding sequences, introns, and Y-untranslated sequences from all three genes. Exons are indicated as hatched boxes, and the identified transcription initiation and polyadenylation sites for PAL1 and PAL2, as well as several prominent restriction sites, are included. Maps are drawn to scale. The restriction fragments cloned for use as gene-specific probes are indicated as a thick solid line underneath the corresponding gene map. The 0.52 kb Hind III fragment from exon 2 of PALl used as a generic PAL gene probe is indicated by a thick broken line. Abbreviations for restriction sites: A, Ava I; B, Barn HI; G, BglII; H, Hind III; N, Nhe I; P, Pst I; Pv, Pvu II; R, Eco RI; S, Sst I; Se, Spe I; Sh, Sph I; V, Eco RV; X, Xba I (Wanner et al. 1995)

The PAL protein consists of 725 amino acids, as deduced from the nucleotide sequence. The general features of the three sequenced regions among PAL proteins of A. thaliana that compared with selected plants species are tabulated in Table ?. Referring to Table ? it can be seen that the nucleotide sequences of the A. thaliana PAL gene reveals that the amino acid coding sequence of PAL1 and PAL2 to be ~ 90 percent identical while only ~70 percent identity similarity between PAL1 and PAL3, and between PAL2 and PAL3 (Wanner et al. 1995).

Table ? Amino acid sequence homology among PAL proteins of A. thaliana that compared with selected

plants species

Gene

AtPAL-1

AtPAL-2

At-PAL-3

Sequence lengh (bp)

AtPAL-1

100 (100)

90 (94.5)

73.3 (84.4)

725

AtPAL-2

90.0 (94.5)

100 (100)

73.3 (83.2)

717

AtPAL-3

73.3(84.4)

73.3(83.2)

100 (100)

694

Parsley

81.8 (90.2)

81.8 (90.0)

74.0(84.2)

716

Bean

80.5 (88.3)

80.2 (88.3)

74.2(84.1)

713

Rice

69.7 (82.8)

69.3 (81.8)

67.0 (80.5)

710

Potato

68.4 (82.8)

69.0 (82.8)

63.5 (82.8)

701

2.6.3 Cinnamate 4-Hydroxylase (C4H) genes in A.thaliana

Cinnamate 4-Hydroxylase (C4H) genes are present in abundant quantities in plants (Mizutani 1987) and catalyses the p-hydroxylation of trans- cinnamic acid to produce p-coumaric acid, in the second most important step of the of the general phenylpropanoid pathway (Fig. 1). The genes encoding for CH4 has been identified by Mizutani et al. (1993) from the purification of the enzyme from Jerusalem artichoke (Teutsch et al. 1993) and mung bean (Mizutani, Ohta & Sato 1997). Teutsch et al. (1993) studied the CH4 gene and found that that CH4 comprises of the CYP73 family of the large group of cytochrome P450 monooxygenases.

Mizutani's group (1997) describes the isolation of the C4H cDNA from A. thaliana and characterization of its expression patterns in response to wounding and light. It was also found that,C4H increases accumulation of phenylpropanoid end-products as well as lignin deposition while the mutant form reversed the reaction. These observations indicate that the C4H gene is has an important role in the development process of A.thaliana (Schilmiller et al. 2009).

It is shown by RNA gel blot analysis, that CH4 is expressed in accordance with PAL1 and other enzymes of the phenylpropanoid pathway, suggesting there is a common transcriptional activation for these genes. Mizutani et al. (1997) further describes that the C4H possess the cis-elements (boxes I', A, and L) at the 5' end of the promoter which are also present in the PAL and 4CL genes (Logemann, Parniske & Hahlbrock 1995). Figure ?? illustrates the structure of the C4H gene in A.thaliana.

Figure ????? The structure of the C4H gene in Arabidopsis and the GUS construct that was used to evaluate the

tissue specificity of C4H expression. (Chapple)

2.7 Gene constructs to over express polyphenols.

As mentioned earlier, all sphagnum species are characterized by a high amount of free endogenous trans sphagnum acid (Rasmussen, Wolfet al 1995). It is well documented (Tutschek 1982 Rasmussen 1996) that sphagnum acid is derived from the phenylpropanoid pathway from phenylalanine. Although the enzyme trans-sphagnum acid synthase has not been isolated remains, the genes coding for the other enzymes, PAL and C4H in the pathway have already have been isolated and subjected to intense study from model plant species Arabidopsis (Cochrane, Davin & Lewis 2004, Ohl et al. 1990, Wanner et al. 1995, Bell-Lelong et al. 1997, Mizutani, Ohta & Sato 1997, Schilmiller et al. 2009). Arabidopsis provides an excellent system for the creation of transgenic plants containing selectable or screenable marker genes regulated by the PAL promoter, or specific dissected c/s-elements, for use in the identification of novel signal pathway mutants that affect PAL promoter activity in trans.

In order to decrease the acceleration of decomposition, we can test the hypothesis that the lower content of decay-inhibiting polyphenols as a result of increasing nitrogen levels accelerates peat decomposition, thereby releasing stored carbon. One way of doing this is to artificially increase the expression of PAL and C4H genes in the pathway leading to polyphenol synthesis.

Primers synthesised to isolate PAL and C4H genes from Sphagnum. These primers will be based on Arabidopsis cloned sequence.

To isolate the C4H gene segment, primers were designed based on the ...

Primer constructed to the Arabidopsis PAL and C4H genes

PCR to amplify up the corresponding Sphagnum homologues

Cloning - plasmid or phage vector.

Northern analysis

Table 1. The peatland comprised of four stages.

Stage Description

SF Swamp Forest seasonally flooded woodland with an understory of shrubs and forest mosses.

FL Fen Lagg poorly minerotrophic peatland with a diverse assemblage of sedges, peat mosses,

and other plant groups.

BM Bog Margin well drained ombrotrophic peatland with low, open tree cover and an understory

of dwarf shrubs and peat mosses.

BP Bog Plateau ombrotrophic peatland with a distinct microtopography of dry hummocks and wet

hollows.

Abbreviations: ABRC, Arabidopsis Biological Resource Center; CAD, cinnamyl alcohol dehydrogenase; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl b-D-thiogalactoside; pkat/ lg, pmoles of substrate converted to product per second per lg protein; ORF, open reading frame; PAL, phenylalanine ammonia lyase; RT-PCR, reverse transcription-polymerase chain reaction; UTR, untranslated region.

The resulting phenolics are often converted into more reactive species by phenol oxidases and

Peroxidises