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Institutionen för fysik, kemi och biologi

Examenarbete

A bioinformatics approach to investigate the

function of non specific lipid transfer proteins in

Arabidopsis thaliana

Muneeswaran Jayachandra Pandiyan

Examensarbetet utfört vid

Linköpings universitet

[2010-06-11]

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Rapporttyp Report category Licentiatavhandling x Examensarbete C-uppsats x D-uppsats Övrig rapport _______________ Språk Language Svenska/Swedish x Engelska/English ________________ Titel Title:

A bioinformatics approach to investigate the function of non specific lipid transfer proteins in

Arabidopsis thaliana.

Författare

Author: Muneeswaran Jayachandra Pandiyan

Sammanfattning

Abstract:

Plant non specific lipid transfer proteins (nsLTPs) enhance in vitro transfer of phospholipids between membranes. Our analysis exploited the large amount of Arabidopsis transcriptome data in public databases to learn more about the function of nsLTPs. The analysis revealed that some nsLTPs are expressed only in roots, some are seed specific, and others are specific for tissues above ground whereas certain nsLTPs show a more general expression pattern. Only few nsLTPs showed a strong up or downregulation after that the Arabidopsis plant had suffered from biotic or abiotic stresses. However, salt, high osmosis and UV-B radiation caused upregulation of some nsLTP genes. Further, when the coexpression pattern of the A.thaliana nsLTPs were investigated, we found that there were several modules of nsLTP genes that showed strong coexpression indicating an involvement in related biological processes. Our finding reveals that the nsLTPs gene was significantly correlated with lipase and peroxidase activity. Hence we concluded that the nsLTPs may play a role in seed germination, signalling and ligning biosynthesis.

ISBN

LITH-IFM-EX—10/2315—SE

__________________________________________________ ISRN

__________________________________________________

Serietitel och serienummer ISSN

Title of series, numbering

Handledare

Supervisor: Johan Edqvist

Ort

Location: Linköping

Datum 2010-06-11

URL för elektronisk version

Nyckelord

Keyword:

Avdelning, Institution

Division, Department

Avdelningen för biologi

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Contents

1 Abstract ... 1

2 List of abbreviations ... 1

3 Introduction ... 1

3.1 Arabidopsis thaliana ... 1

3.2 Plant non-specific lipid transfer proteins (nsLTPs)... 2

3.3 Arabidopsis Microarray data of gene expression ... 3

4 Materials and methods ... 3

4.1 AtGenExpress micro array data sets and AtGenExpress visualization tool... 3

4.2 Genevestigator ... 3

4.3 Bio Array Resource ... 4

4.4 CORNET ... 4

4.5 BiNGO 2.3 ... 5

5 Results ... 5

5.1 Analysis of tissue distribution of the Arabidopsis nsLTPs genes ... 5

5.2 Regulatory analysis of the Arabidopsis nsLTPs Gene ... 9

5.3 Coexpression analysis of the Arabidopsis nsLTP genes ... 10

5.4 Construction of networks of coexpressed genes ... 11

5.5 Enrichment of GO terms in the expression networks ... 11

6 Discussion ... 13

6.1 Coexpression analysis ... 14

6.2 Future prospect of analysis of specific function... 15

6.3 Conclusion ... 15

7 Acknowledgements ... 15

8 References ... 15

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1 Abstract

Plant non specific lipid transfer proteins (nsLTPs) enhance in vitro transfer of phospholipids between membranes. Our analysis exploited the large amount of

Arabidopsis transcriptome data in public databases to learn more about the function of

nsLTPs. The analysis revealed that some nsLTPs are expressed only in roots, some are seed specific, and others are specific for tissues above ground whereas certain nsLTPs show a more general expression pattern. Only few nsLTPs showed a strong up or downregulation after that the Arabidopsis plant had suffered from biotic or abiotic stresses. However, salt, high osmosis and UV-B radiation caused upregulation of some nsLTP genes. Further, when the coexpression pattern of the A.thaliana nsLTPs were investigated, we found that there were several modules of nsLTP genes that showed strong coexpression indicating an involvement in related biological processes. Our finding reveals that the nsLTPs gene was significantly correlated with lipase and peroxidase activity. Hence we concluded that the nsLTPs may play a role in seed germination, signalling and ligning biosynthesis.

Keywords: nsLTPs, Arabidopsis, tissue, stress, regulation.

2 List of abbreviations

ABA - abscisic acid coex - coexpression

EXLs - extracellular lipase GPI- glycosylphosphatidylinostol GO - Gene Ontology

Nacl - Sodium chloride

nsLTPs - non specific lipid transfer proteins

THLTP - Tamarix hispida lipid transfer protein

3 Introduction

3.1 Arabidopsis thaliana

Arabidopsis thaliana is a plant that is frequently used in molecular genetics. Arabidopsis

or thale cress is a flowering plant of mustard family. It is one of the most widely used model organisms of plant geneticists and molecular biologists. It has several traits that make it a useful when investigating the genetics, cell biology, and molecular biology of flowering plants. Arabidopsis is small and grow easily, so it allows researchers to cultivate a large number of plants in the laboratory spaces and also it has one of the smallest genome in the plant kingdom. Its genome is only about 125 million base pairs. Another important property of Arabidopsis is that it is rather uncomplicated to transform with foreign DNA. These features of the Arabidopsis plants are of great help in research aiming at determining the function of genes or physical interactions between genes or their gene products. Recently, the complete Arabidopsis genome was analyzed and showed to contain approximately 25 000 genes. As a model organism Arabidopsis presents excellent opportunities to provide key insights to study the gene expression.

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3.2 Plant non-specific lipid transfer proteins (nsLTPs)

Non-specific lipid transfer proteins (nsLTPs) are defined by their ability to transfer lipids between membranes in vitro (Thoma et al. 1994). NsLTPs have been isolated and described from various flowering plants including spinach, maize, castor bean, and wheat. The nsLTPS are encoded by large gene families with 50-100 members in many flowering plants (Boutrot et al. 2008). NsLTPs are characterised by four α-helices which are stabilized by four conserved disulfide bridges formed by an eight cysteine motif (8CM) through the general form of C-Xn-C-Xn-CC-Xn-CXC-Xn-C-C. The nsLTPs are structurally marked by the presence of a tunnel-like hydrophobic cavity, which is properly designed for the binding of hydrophobic ligands, such as lipids (Kader et al. 1984: Lee et al. 1998; Lascombe et al. 2008). Almost all nsLTPs are holding an N-terminal signal peptide to direct the protein to the apoplastic space. Some also have a sequence motif for the post-translational addition of a glycosylphosphatidylinositol (GPI)-anchor that may attach the proteins to the plasma membrane (Lee et al. 2009). On the basis of molecular masses, plant nsLTPs have been classified into two types: type I consist of approximately 90 amino acids and type II have 70 amino acids (Douliez et al. 2000). NsLTP type G consists of nsLTPs with a GPI-anchor. NsLTPs are involved in a wide range of biological processes, but an exact biological role is not clearly understood. The nsLTPs bind to sterol molecules to trigger the plant defense response by interacting with a receptor at the plant plasma membrane (Wang et al. 2007; Carvalho and Gomes, 2007). Therefore the nsLTPs may be involved in the plant defense against viral, bacterial and fungal pathogens (Molina et al. 1993; Gomes et al. 2003). Different member of the nsLTP family have also been identified as allergens in food and pollen (Salcedo et al. 2004). NsLTPs may also play a significant role in the formation of protective hydrophobic layers on the surface of the plant aerial organs possibly by stimulating the transfer of hydrophobic compounds to the extracellular environment (Trevino et al. 1998; Lee et al. 2009).

Various ligand binding specificities and different expression patterns make it difficult to draw any conclusions about the functions of nsLTPs in plants. In general, the expression is detectable during the early development stage of the plant (Sterk et al. 1991; Thoma et al. 1994; Vroemen et al. 1996). A different member of the nsLTP genes shows the various expressions in the individual tissues in Arabidopsis and also observed in other species such as tomato (Lycopersicon esculentum) (Trevino and O’ Connell, 1998; Clark and Bohnert, 1999). As nsLTPs are extracellular, it is unlikely that the functional roles of these proteins are intracellular but the nsLTPs are mainly expressed in the epidermis tissue particularly in the aerial organ (Kader, 1997). The Arabidopsis Ltp1 is expressed

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3.3 Arabidopsis Microarray data of gene expression

The increase in the number of genome-wide data, mediates the development of advanced system biology. Further microarray experiments for different model species provide detailed description of the expression of genes in specific tissues and in response to different stimuli. One useful tool is the database AtGenExpress that consists of more than 500 microarray data sets from experiments with Arabidopsis during different developmental stages, during many different abiotic and biotic stress conditions and in response to various stimulus The AtGenExpress experiments are mainly based on the Affymetrix ATH1 GeneChip (Kilian et al. 2007; Goda et al. 2008). Bioinformatic databases and tools such as the Bio-Array Resource (BAR Toufighi et al. 2005), Genevestigator (Zimmermann et al. 2004), ACT (Manifield et al. 2006), ATCOECIS (Vandepoele et al. 2009), ATTED-II (Obayashi et al. 2007 2009), CressExpress (Srinivasasainagendra et al. 2008), CSB.DB (Steinhauser et al. 2004), PRIME (Akiyama et al. 2008), CORNET (De Bodt et al. 2010) and Plant Gene Expression Database (Horan et al. 2008) are used for visualising the micro array data or for detecting relationships between genes based on their expression. Another very useful tool is the Gene Ontology (GO), which provides a controlled vocabulary of terms for annotations of gene function. By combining gene specific microarray data, calculations of coexpression, construction of expression networks and investigation of enriched GO terms, it may be possible to obtain clues to function of genes (Ma et al. 2007).

The main goal of this study were 1) to analyze the expression profile of the Arabidopsis nsLTPs genes under different response 2) to obtain the Arabidopsis nsLTPs genes coexpression network 3) to employ GO enrichment analysis to check the biological functional module in the lipid transport in the Arabidopsis.

4 Materials and methods

The nsLTP genes used in this investigation are listed in Appendix.

4.1 AtGenExpress micro array data sets and AtGenExpress visualization tool

Microarray is defined as rectangular slide by stretches of DNA which is chemically bonded (Orlando et al. 2009). The Affymetrix® Arabidopsis ATH1 gene chip have a piece

of DNA known as probes correspond to 24,000 protein-encoding genes. By using this technology, the AtGenExpress data provide the detailed description of nsLTP genes under different environmental abiotic stress conditions including heat, cold, drought, salt, high osmolarity, UV-B light and wounding (Kilian et al. 2007).

The AtGenExpress stress set data was visualized using the AtGenExpress Visualization tools (http://jsp.weigelworld.org/expviz/expviz.jsp). The nsLTP genes were defined as stress induced when the threshold mean-normalization value was > 5.

4.2 Genevestigator

Genevestigator (https://www.genevestigator.com/gv/index.jsp ; Zimmermann et al. 2004) is another tool to visualize the microarray from the AtGen Express data sets. In this study we used Genevestigator on the AtGenExpress data sets to visualize the tissue specific expression of the nsLTPs. Hence, we present our output in heat-map format from the

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Meta-profile analysis tool based on screening of the ATH1 22k array source for maximum expression categorised by Anatomy. The average expression level corresponding to the anatomy structure is noted which is indicated by variation of colour. The data is plotted against the tree of anatomical category.

4.3 Bio Array Resource

To obtain the functional context from the expression pattern of nsLTP genes, it was investigated how the expression of the stress induced nsLTPs gene of interest correlated to the expression of other genes. We calculated Pearson correlation coefficients (R) from available gene correlator functions such as the Expression Angler of the Botany Array Resourcein the University of Toronto (http://bar.utoronto.ca/; Toufighi et al. 2005). The calculations are based on the AtGenExpress data sets. At the Expression Angler, the data are categorized into four groups: tissue, hormone, stress, and pathogen for samples collectedfrom various tissues and developmental stages, after hormonetreatments, after abiotic stresses or biotic stresses, respectively. We screen the stress data for the top 25 hits showing the strongest correlation to the nsLTP used as query gene of each gene. R > 0.7 is normally defined as a rule-of-thumb threshold for true correlation and used in various analyses (Lee et al. 2004; Ren et al. 2005).

4.4 CORNET

Cornet (https://bioinformatics.psb.ugent.be/cornet/main/tool ; De Bodt et al. 2010) is a user friendly database allows to facilitating the coexpression network analysis through the multitude of search options. The calculations in Cornet were based on the AtGenExpress data sets. We used predefined expression data set over the global expression compendium which indicates that the complete AtGenExpress data set is included in the calculations. To measure the similarity in the expression profiles, we set the Pearson correlation coefficient to R>0. We calculated the expression correlation in “query gene(s) with neighbours” (Figure 1) using multiple nsLTP genes as query genes. The query genes corresponded to nsLTPs showing strong coexpression in the Expression Angler analysis. In addition, the mean value for correlation coefficient for the AtGenExpress compendium that met the chosen threshold is reported (Figure 7). Result was displayed in text output as well as in Cytoscape. Cytoscape is a tool for visualizing and editing the constructed gene interaction networks.

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Figure 1 Coexpression analysis: Interaction of query gene(s) with neighbour genes.

4.5 BiNGO 2.3

We assessed the overrepresentation of Gene Ontology (GO) terms in interaction networks constructed with Cornet. We selected the BiNGO plugin in Cytoscape to calculate and visualize the enrichment of significant GO terms. We had chosen the statistical test (hypergeometric test) and Benjamini & Hochberg's FDR correction (multiple correction factors). We set the p value of 0.05 to represent significant GO term in the networks. Finally, visualization of significantly enriched GO term categories was obtained in Cytoscape. The list of significantly overrepresented GO term was stored in (.bgo) format.

5 Results

Previously, 112 genes that probably encode nsLTPs were identified in Arabidopsis (Boutrot et al. 2008). The genes that we have used in this report are listed in Appendix I.

5.1 Analysis of tissue distribution of the Arabidopsis nsLTPs genes

To learn about the tissues specific expression of Arabidopsis nsLTPs, we explored the tissue set of the Arabidopsis microarray experiment AtGenExpress. Genevestigator was used for visualizing the micro array data. The expression pattern of nsLTP Type G are shown in Figure 2 whereas the expression of the remaining nsLTP types is shown in Figure 4.

The graphs in Figures 2 and 3 show the expression in the heap map format. The scale is linear. The colour variation indicates the expression level of the genes, where the darker colours indicate higher expression levels. At least four of the nsLTPs Types G (At2g13820, At1g62790, At2g44300, At3g22600) are expressed mostly in the root while the few type G are specific for tissues above the ground. As shown in Figures 2 and 3, the type G genes have their expression maximum either in flowers, roots or seeds. Other type of nsLTPs genes specific to the seed and at least two seems to be expressed both above and below the ground. It is interesting that none of the nsLTPs analysed in Figures 4 and 5 show a root specific pattern. Further, several of the nsLTPs in Figure 4 are highly expressed in xylem and there are also some which are abundant in stem. Thus, it seems that the Type G is important in different tissues when compared to the other type I-V.

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Figure 2 . The tissue specific expression pattern of Arabidopsis nsLTP Type G genes. A maximum expression is indicated by dark blue colour. Lighter colours indicate lower expression levels.

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Figure 3. Tissue distribution of Arabidopsis nsLTP typ G genes. It is shown in which tissue a given nsLTP Type G has its highest expression level. a-Flower, b-Seed, C-Root, D-Rosette.

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Figure 4.Expression pattern of the nsLTP gene. Gene expression pattern of Arabidopsis nsLTPs other than type G. Maximum expression is indicated by dark blue colour.

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Figure 5. NsLTPs gene in the different tissue of Arabidopsis. The figure show in which the tissue a certain putative nsLTPs other than type G is most abundant. a-Flower, b-Seed, c-Root, d-Rosette.

5.2 Regulatory analysis of the Arabidopsis nsLTPs Gene

AtGenExpress data set contains the information from experiments of the Arabidopsis in responses to different abiotic stresses. We used AtGenExpressr to visualize the stress response in order to identify the stress conditions that upregulate or downregulate the expression of Arabidopsis nsLTPs. The results are shown in the Figures 6 and 7. Rather few nsLTP genes showed a clear response to any of the tested stress conditions. However, some genes were upregulated as a consequence of UV-B, salt and osmotic stress. Other abiotic and biotic stress conditions did not influence the expression of the

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Figure 6.Stress induced up-regulation of Arabidopsis nsLTP expression in root tissues.

Figure 7. Stress induced up-regulation of Arabidopsis nsLTP expression in seedlings.

5.3 Coexpression analysis of the Arabidopsis nsLTP genes

To establish a functional context of the Arabidopsis nsLTPs, it was analysed whether the expression of the stress induced nsLTPs is correlated to other genes. A strong coexpresssion of two genes may indicate that the genes are involved in the same biological process. The Expression Angler of the Bio Array Resource (Toufighi et al. 2005) was used for the calculation of Pearson correlation coefficient (R) from the stress set of the AtGenExpress microarray experiment. The stress induced nsLTPs were selected as query genes. We obtained top lists with the 25 genes showing the strongest correlation to each stress induced nsLTP. In the top lists we manually identified other nsLTPs that, thus, showed significant coexpression to the query nsLTPs (Figure 8).

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5.4 Construction of networks of coexpressed genes

To further investigate the biological context of nsLTPs we used the bioinfomatics tool CORNET (De Bodt et al. 2010) to build networks of genes coexpressed with Arabidopsis nsLTPs. Once again, the calculations were based on the AtGenExpress micro array data set. The identified nsLTP expression modules (Figure 8) were used as query genes. CORNET was set to identify query genes with neighbours in order to construct a network of genes with expression pattern correlating to the multiple genes in each module. Graphical visualizations of the obtained networks for all four modules are shown in Figure 9.

Figure 8.Coexpression pattern of Arabidospis nsLTP genes calculated by the Expression angler at Botany of Array (BAR) database in University of Toronto. Each gene are represented by a specific colour.

5.5 Enrichment of GO terms in the expression networks

Numerous plugins can be exploited to explore the network; we enabled BiNGO v2.3 in Cytoscape to perform GO enrichment analysis to improve our understanding of functional context of each module of nsLTPs.

Table 1 lists the GO molecular function and biological process that were significantly over-represented in each module. Network visualization for overrepresented GO Molecular function is shown in Figure 10. In Module I; the lipase activity shows the significant molecular function while the biological process expressed significant GO term towards the lipid transport. Module II was strongly correalted to peroxidase activity and oxidative stress. In module III, the phenylpropanoid pathway was overrepresented in both GO term, but the module IV did not show any significant enrichment of GO term. The result may suggest an involvement for these nsLTPs in signaling, seed germination and ligning formation.

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Figure 9.Visualization of network. Module I (4)*; Module II (4)*; Module III (5)*; Module IV (4)*. * Number of query genes used in the CORNET database.

Table 1.Comparison of significant enrichment GO term for nsLTPs.

Modules GO term p-value GO term p-value Biological Process

Molecular Function

Module I *

Mol Funct lipid

binding 1.19E-18

lipid transport 2.98E-10 lipid transport 1.81E-09

* lipase activity 2.01E-05

Module II * oxidoreductase activity, acting on peroxide as acceptor 2.16E-13

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phenylpropanoid

metabolic process 1.84E-03

phenylpropanoid

metabolic process 1.48E-05 *Not applicable

Figure 10.Significantly over-represented GO Molecular Function detected in Module I, II, III. Each colored circle represents an over-represented GO term. The color scale indicates the p value of the over-represented. GO term. An arrow from GO term A to Go term B indicates that A is the parent of B. Module IV doen’t show significantly over-represented GO term.Yellow colour nodes indicate the significant level of GO term and orange colour indicates the more significant overrepresented GO term.

6 Discussion

In this report, we have explored available Arabidopsis micro array data in order to find clues to the function of individual members of the nsLTP gene family. One of our observations is that many nsLTPs show a tissue specific expression pattern. For instance, some of the nsLTPs are highly expressed in seeds, some in roots, some in flowers, whereas a few are most abundant in the xylem. This indicates the gene families have expanded to carry out many different functions in the plant. Interestingly, only members of the Type G family showed a root specific pattern. This was rather surprising since the

Arabidopsis LTPG, which is an nsLTP TypeG, previously was shown to be involved in

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(DeBono et al. 2009; Lee et al. 2009). Further, other Arabidopsis nsLTP Type G showed high expression levels in floral organs, such as stamen and stigma. This suggests that several nsLTP Type G proteins play important roles in reproduction in flowering plants. Seemingly, our data suggests that the nsLTP Type G are not only important for the formation of the cuticle on aerial surfaces, they are also involved in many other processes in a flowering plant.

Certain nsLTP genes induced through abiotic stress, but this induction is seen in the plant tissues do not shows the expression of those genes. Here we showed the induction of certain nsLTP genes on various stresses in the root and seedlings. Few reports already showed that the gene encoding an LTP like protein in tomato also induced in stem by Nacl, mannitol and ABA treatment (Torres-Schumann et al. 1992). In barley, a gene which is partially homologous to LTP induced drought (Plant et al. 1991) and the other genes also induced in the root and stem of drought stressed barley (White et al. 1994). Induction of salt stress or ABA treatment which results in the increase in LTP mRNA levels in germination seeds (Soufleri et al. 1996; Vignols et al. 1997). The findings from Wang et al. (2009) showed that ThLTP (Tamarix hispida) expression represent an adaptive response to abiotic stresses and involved in abiotic stress tolerance. THLTPs was expressed (> 2-fold) of ABA treatment in root, leaf and stem.

6.1 Coexpression analysis

The main approach used in this study is to develop coexpression network for stress induced nsLTP genes. We present the system level understanding of gene module that facilitates the different biological function used to carry out particular biological process (Linyong et al. 2009). Coexpress of gene network pattern generated by visual/manual and graphical format in this study may be showed the similar and various biological functions. Hence, the coexpression network analysis may find the regulatory function and metabolic pathway of gene (Hirai et al. 2004).

In each module, there are several GO terms of these; few of the GO terms are significantly over-represented. In the gene network constructed from module I, the GO term analysis revealed that the term lipase activity was significantly enriched. This results suggests that the nsLTPs in module I are expressed at the same time as lipases. Lipases areimplicated in disease resistance, signalling, seed germination and pollen maturation in plants. In flowering plant, pollination starts with hydration of desiccated pollen grains on the stigma. Both lipid and protein is important for hydration. Arabidopsis pollen coat is important for mediating the contact between pollen and stigma, facilitate hydration (Updegraff et al. 2009). Arabidopsis Pollen coat contain extracellular lipase (EXLs). EXL4 and EXL6 are available in the pollen coat in which EXL4 is efficiently important for pollen hydration (Mayfield et al. 2001). Studies from Updegraff et al (2009) shows

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H2O2 and in the defense against pathogen (Tognolli et al. 2002). During wound condition, the expression of peroxidase repairs the damaged tissue and also stop defense mechanism against pathogen attacks (Tognolli et al. 2002). Hence, the class III peroxidases are well recognized for being involved in the defense and stress regulatory mechanisms while they are also involved in the lignifications. Another significant GO term corresponding to modules III was phenylpropanoid pathway which helps plant towards biotic and abiotic stresses and they are act as key mediators for plant resistance towards pest. (La camera et al. 2004). Small member of gene family in arabidopsis involve in the phenylpropanoid pathway and also in the lignin formation (Hamberger et al. 2004). Recent report suggested that the phenylpropanoid not only common to lignin and flavonoid biosynthesis, they also pertain to different aromatic metabolites.

6.2 Future prospect of analysis of specific function

In future, the increased number of the gene expression data, biochemistry and biological process of individual genes is essential to understand the regulation, pathway of the key functions of the genes. Different approaches will lead to the better understanding of biological process which includes the analysis of promoters and the analysis of knock-out mutants for nsLTP genes. The combination of those approaches may provide a lot of information from microarray analysis of particular gene respect to the different stress response, developmental stage and stimuli.

6.3 Conclusion

We used coexpression network approach to study the functional modules of genes from the large microarray databases. Our result suggests that nsLTPs are coexpressed with lipase activity and peroxidase. This suggests that possible roles for nsLTPs in signalling, seed germination and lignin biosynthesis.

7 Acknowledgements

I am heartily thankful to my supervisor, Johan Edqvist, whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the subject.

Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the project.

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9 Appendix

The following lists of genes are used for studies (Boutrot et al. 2008).

Table 1. NsLtp genes identified in the Arabidopsis thaliana genome nsLtp gene locus/model TypeI AtLtpI.1 At2g15050.2 AtLtpI.2 At2g15325.1 AtLtpI.3 At2g18370.1 AtLtpI.4 At2g38530.1 AtLtpI.5 At2g38540.1 AtLtpI.6 At3g08770.1 AtLtpI.7 At3g51590.1 AtLtpI.8 At3g51600.1 AtLtpI.9 At4g33355.1 AtLtpI.10 At5g01870.1 AtLtpI.11 At5g59310.1 AtLtpI.12 At5g59320.1 TypeII AtLtpII.1 At1g43665 b AtLtpII.2 At1g43666.1 AtLtpII.3 At1g43667.1 AtLtpII.4 At1g48750.1 AtLtpII.5 At1g66850.1 AtLtpII.6 At1g73780.1 AtLtpII.7 At2g14846.1 AtLtpII.8 At3g12545 c AtLtpII.9 At3g18280.1 AtLtpII.10 At3g29105 d AtLtpII.11 At3g57310.1 AtLtpII.12 At5g38160.1 AtLtpII.13 At5g38170.1 AtLtpII.14 At5g38180.1 AtLtpII.15 At5g38195.1 TypeIII AtLtpIII.1 At5g07230.1 AtLtpIII.2 At5g52160.1 AtLtpIII.3 At5g62080.1

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TypeIV AtLtpIV.1 At5g48485.1 AtLtpIV.2 At5g48490.1 AtLtpIV.3 At5g55410.1 AtLtpIV.4 At5g55450.1 |AtLtpIV.5 At5g55460.1 TypeV AtLtpV.1 At2g37870.1 AtLtpV.2 At3g53980.1 AtLtpV.3 At5g05960.1 TypeVI AtLtpVI.1 At1g32280.1 AtLtpVI.2 At4g30880.1 AtLtpVI.3 At4g33550 e AtLtpVI.4 At5g56480.1 TypeVIII AtLtpVIII.1 At1g70250 f Type IX AtLtpIX.1 At3g07450.1 AtLtpIX.2 At3g52130.1 nsLTPY AtLtpY.1 At1g52415 g AtLtpY.2 At1g64235 h AtLtpY.3 At4g08530 i AtLtpY.4 At4g28395 j b

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h

annotations curated (strand: +1; exon 1 start: 23839912, end: 23840250; exon 2 start: 23840828, end: 23840845).

i

annotations curated (strand: +1; exon start: 5421971, end: 5422352). j

annotations curated (strand: +1; exon 1 start: 14044281, end: 14044490; exon 2 start: 14044565, end: 14044734; exon 3 start: 14044856, end: 14044898).

Table 2. Arabidopsis nsLTPs Type G genes

Glycosylphosphatidylinositol-anchored proteins At1g03103, At1g05450, At1g18280, At1g27950, At1g36150, At1g55260, At1g62790, At1g73550, At1g73560, At1g73890, At2g13820, At2g27130, At2g44290, At2g44300, At2g48130, At2g48140, At3g22570, At3g22580, At3g22600, At3g22620, At3g43720, At3g58550, At4g08670, At4g12360, At4g14805, At4g14815, At4g22630, At4g22666, At5g09370, At5g13900, At5g64080

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