Content
1 Abstract…………………………….………………........................
2 List of abbreviations ………………………………………………
3 Introduction………………………………..………………………
4 Material and Methods………………….……….…………………
4.1 Cytoscape………………………………………................
4.2 AtGenExpress Visualization tool……………………….…
4.4 Bio Array Resource…………………………………..
4.5 Cytoscape………………………………………………..
4.6 Bingo 2.3…………………………………………………….
5 Results……………………………………………………………...
5.1 Analysis of tissue distribution of the Arabidopsis nsLTPs gene
5.2 Regulatory analysis of the Arabidopsis nsLTPs gene
5.3 Co-expression analysis of the Arabidopsis nsLTP genes
6 Discussion………………………………………………………….
6.1 Anatomical tissue-Genevestigator ………………………………
6.2 Abiotic stress-AtGenExpress…………………………………
6.3 Co-expression analysis…………………………………………
6.4 Conclusion.....................................................................................
7 Acknowledgements………………………………………………..
8 References………………………………………………………….
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1 Abstract
Plant non specific lipid transfer proteins (nsLTPs) can involve in vitro transfer of
phospholipids between the membranes. Hence they were thought to be take part in the
regulation of intracellular fatty acid pools (Kader, 1996). Our analysis includes the expression
pattern of the Arabidopsis nsLTPs genes and also finding out the biological process in the coexpression network of nsLTPs through the different microarray databases. Anatomy-tissue
studies of nsLTP genes expression were observed significantly in the seed and also other part
of the plant through the Genevestigator (Zimmermann et al. 2004). AtGenExpress database
analysis abiotic stress induced gene expression pattern for nsLTP genes for certain threshold
value (Kilian et al. 2007). Furthermore, co-expression pattern of those stress induced nsLTP
genes were generated from the Expression Angler of the Bio-Array Resource (BAR; Toufighi
et al. 2005) and CORNET (Stefanie et al. 2010). Hence co-expression network analysis
indicates that the biological process such as phenylpropanoid metabolic process, oxidative
stress, defense response might play a role in the lipid transport. Therefore, co-express network
analysis identified the biological process which can serve as functional annotation for
studying the nsLTPs regulation. In this conclusion, analysis of co-expression pattern for
Arabidopsis stress induced nsLTP genes may involve in the oxidative stress during the
biological process of lipid transport. This network analysis revels new insight for the
functional study of gene.
Keywords:
Keywords: nsLTPs, Arabidopsis, co-express, stress, microarray, network.
2 List of abbreviations
GPI- glycosylphosphatidylinositol
GO – Gene Ontology
nsLTPs – non specific lipid transfer protein
3 Introduction
Plant non-specific lipid proteins were firstly identified from the spinach leaves and hence
named for their propery to mediate in vitro transfer of Phospholipids between membranes
(Kader et al. 1984). NsLTPs are widely distributed in the plant kingdom and forms multigenic
families of related proteins (Boutrot et al. 2008). NsLTPs are characterised by four α-helices
which they are stabilized by four conserved disulfide bridges fashioned by an eight cysteine
motif (8CM) through the general form of C-Xn-C-Xn-CC-Xn-CXC-Xn-C-C. The nsLTPs are
strcturally marked by the presence of tunnel-like hydrophobic cavity, which is involved in the
transferring of various lipid molecules between lipid bilayers in vitro (Kader et al. 1984).
Approximately, all nsLTPs are holding an N-terminal signal peptide to tolerate to the
apoplastic space and some have a sequence motif for the post-translational addition of a
glycosylphosphatidylinositol (GPI)-anchor (Lee et al. 2009). On the basis of molecular
masses, plant nsLTPs were classified into two types: type I consist of exactly 90 amino acids
and type II have 70 amino acids (Douliez et al., 2000). Type I and II nsLTPs were represent a
structurally related protein family (Boutrot et al., 2008). There are a number of anther specific
protein which is homology to plant nsLTPs comes under the third type differs from the other
two types by the number of amino acid residues (Boutrot et al. 2005).
NsLTPs are involved in a wide range of biological process, but an exact biological role in
vitro 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 (Gomes et al, 2003; Molina et al, 1993). Low
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molecular mass cysteine-rich proteins possess intrinsic antimicrobial properties tends to
involve in the plant defense mechanism (Broekaert et al, 1997; Garcia-Olmedo et al. 1998).
NsLTPs play a significant role in the formation of a protective hydrophobic layer over the
plant surface which helps to transfer hydrophobic ligands in the presence of extracellular
environment (Lee et al. 2009; Trevino et al. 1998).
Various biochemical action and different expression patterns make it difficult to draw any
conclusions about the functions of the nsLTPs in the plant. Hence it shows that the individual
nsLTP genes have different functional roles in different tissues during development stage
(Beatrix et al. 2002). A different member of the nsLTP genes shows the different expression
in the individual tissues in Arabidopsis and tomato (Lycopersicon esculentum) (Trevino and
O’ Connell, 1998; Clark and Bohnert, 1999). Expression is generally detectable during the
early development stage of the plant (Sterk et al. 1991; Thoma et al. 1994; Vroemen et al.
1996). NsLTPs are mainly expressed in the epidermis tissue particularly in the aerial organ
(Kader, 1997). The Arabidopsis Ltp1 is epidermis-specific and it is expressed mainly in the
flowers and embryos and also it is highly expressed in the leaves. Ltp2 and Ltp3 are also
expressed in the cell layers internal to the flower and embryo organs except the leaves (Clark
et al. 1999). A few nsLTPs expressed highly in the endosperm of germinating seeds and may
be involve in the recycling of endosperm lipids or as protease inhibitors, thus protecting the
cotyledons from protease released from the endosperm during programmed cell death
(Edqvist and Farbos, 2002 and Edqvist, 2003).
The increase in the number of genome-wide data, which explain the functional properties of
the genes, mediates the development of advance system biology. Applications of microarray
experiment for different model species provide the detailed description of the expression of
genes in specific tissues and in response to different stimuli. Genome wide expression profile
created for individual organisms like AtGenExpress consists of more than 500 data set from
experiments with Arabidopsis (Arabidopsis thaliana) development and response to various
stimulus based on the Affymetrix ATH1 GeneChip (Schmid et al. 2005; 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) and Plant Gene Expression Database (Horan et al, 2008) also useful in
identifying the similarity between genes based on their expression. The Gene Ontology (GO)
annotation in combination with a statistical test is widely used to find the functional
enrichment. Some studies identified the highly connected cluster from the Arabidopsis gene
network group associated with the biological pathways and cold stress (Ma et al. 2007).
Using user friendly databases, expression pattern of a gene in tissue under different condition
are to be obtained. These may be further used in analysis of multiple biological functions. 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 co-expression
network 3) to employ GO enrichment analysis to check the biological functional module in
the lipid transport in the Arabidopsis.
4 Materials and methods
4.1 Genevestigator:
Genevestigator (Zimmermann et al. 2004) have the meta-profile tool sets which allows users
to find the expression level in certain tissues like roots, leaves or flower. Anatomy tool was
divided into six different main groups such as callus, cell suspension, seedling, inflorescence,
rosette, and roots and the corresponding subgroups. These category cover all tissues that can
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currently be isolated for expression analysis, but can easily be extended as tissue and cell
separation techniques become more precise (Birnbaum et al. 2003).
Arabidopsis Type G and other putative nsLTP genes are viewed in the anatomy meta-profile
tool. Expression pattern are display in the heat map and the scale is in linear. The average
expression level corresponding to the anatomy structure is noted which is indicated by
variation of color. The data is plotted against the tree of anatomical category.
4.2 AtGenExpress Visualization tools:
AtGenExpress develop a wide range of Arabidopsis thaliana gene expression by using the
Affymetrix ATH1 microarray (Kilian et al. 2007). A database consists of experimental
condition of 1094 ATH1 microarray. Different abiotic stress such as wounding, heat, cold,
salt, osmotic, oxidative and UV light is analysed for arabidopsis type G and putative nsLTP
genes. Mean normalization value is predict for up regulate genes according to each stress in
particular time and tissue. The value is the mean normalization which is determined by set the
threshold value (i.e. the value more than 5).
4.3 Bio Array Resource:
Expression angling tool is used to identify the co-regulated genes for stress induced nsLTP
genes (Toufighi et al. 2005). Further, up regulated genes are subject to co-regulation analysis.
The tool which calculates the Pearson correlation across the co-expression of genes by set of
expression levels across all the experiments. If the range from 1 shows the perfect correlation
and -1 is anti-correlation and zero which means that there is no correlation (Toufighi et al.
2005). Each up regulated gene undergo the AtGenExpress Stress and Tissue set for coexpression test, the data which return the r-value for top 25 hits and there is the default r-value
cutoff range from 0.75 to 1.00.
4.4 Cytoscape:
It is the bioinformatics software platform to visualize the biological pathway and interaction
of molecules and link this interaction with gene expression. Cytoscape_v2.6.3 is used to from
the co-express network. Network laywork layout display in yFile organic algorithm.
4.5 BiNGO 2.3:
The Cytpscape plugin BiNGO 2.3 is used to visualize GO term enrichment (Maere 2005).
Bonferroni Family-Wise Error Rate (FWER) correction is used to control the false positive
rate. If GO term in the network showed FWER corrected p value of 0.05, then the biological
process is significantly enriched.
.
5 Results
5.1 Analysis of tissue distribution of the Arabidopsis nsLTPs genes:
Studies from the locus annotation and protein domain identified 112 loci that probably encode
nsLTPs (Boutrot et al., 2008). Figure 1 and 2 show the expressions of the type G encode GPIAPS and putative nsLTPs gene in different tissue of the Arabidopsis. Right side image in the
figure 1 and 2 explain the tissue distributions level from the Genevestigator’s anatomy tool.
The graph displays in the heap map format and the scale is linear. The maximum expression
is indicted by dark blur colour. The colour variation indicates the expression level of the
genes. NsLTP encode GPI-APS are highly expressed in the flower, root and seed but not in
the rosette while the other putative nsLTPs gene expressed maximum in the flower, seed,
rosette and root. So, there are some genes expressed in almost all the tissue which include the
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subcategory of flower, seed, root and rosette while the other had specific expression of
pattern.
Figure 1 Tissue distribution of the Arabidopsis type G. A. 23 out of the 31 loci shows the gene
expression in different tissue from the Gevevestigator's anatomy tool; B. Picture explains the
presence of nsLtp type G in the flower, seed, and root. Seedling is not display in the picture.
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Figure 2 Expression of nsLTPs gene in the Arabidopsis. A. 48 out of the 112 loci encode
putative nsLTPs gene shows the gene expression in the different tissue from the
Genevestigator's tool. B. The presence of nsLTPs gene in the flower, seed, rosette and root.
5.2 Regulatory analysis of the Arabidopsis nsLTPs Gene:
AtGenExpress consortium which contains the data set from experiments inspects the
Arabidopsis responses to different stress. Hence abiotic stress regulations are scan in
AtGenExpress Visualization tool. The considerable up regulation of nsLTPs gene response to
stress in the root and seedling are obtained in the form of mean-normalization scale. Here, the
up regulations of the nsLTPs gene response to UV-B, osmotic, salt stress were illustrated in
the Figure 3 and 4. Results are quite interesting that typeI nsLtp gene (At5g59310) expressed
the maximum osmotic and salt stress in the seedling in 24h while the typeV nsLtp gene
(At2g37870) shows the maximum up regulation of osmotic and salt stress in the root in 24h.
Threshold value on mean normalization scale was set at 5 to distinguish up or down
regulation of nsLTP genes. Consequently the other genes did not show significant up
regulation.
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Figure 3 Abiotic stress up regulation of the Arabidopsis nsLTPs gene in the root.
Figure 4 Abiotic stress up regulation of the Arabidopsis nsLTPs gene in the seedling.
5.3 Co-expression analysis of the Arabidopsis nsLTP genes:
Bio Array Resource (BAR) expression profile program has the novel tool known as
Expression angler permits genes showing similar expression or response from the selected
database (Toufighi et al. 2005). Analysis of genes may neither under vent up or down
regulation may not lead to productive information. Hence, the genes that only under vent up
regulation were analyzed. Up regulated nsLtps genes in the root and seedling from the Figure
3 and 4 undergoes Expression angler tool for similar gene expression analysis. Correlated
gene is predicted based on Pearson correlation coefficient (r-value) cutoffs. If the highest the
value is 1, then the two vectors are in the perfect match. Based on Pearson correlation
coefficient and also visualization of annotation, the co-expression pattern for AtGenExpress
stress and AtGenExpress tissue set is achieved as shown in Figure 5 and 6. Most of the genes
in the AtGenExpress stress set show the similar gene expression and also they are correlated
with pairwise/neighbor genes/each other.
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Figure 5 Coexpress pattern of the Arabidospis nsLTPs gene from the novel tool, Expression
angler (Bio Array Resource).
Co-expressed genes were collected for only up regulated genes, but co-express genes have
similar functions. This is emphasized by r>6 which means that they are corregulate with each
other. The colour network has been formed by visual and manually. This colour network has
been supplemented by bioinformatics tool called CORNET. The simple co-expression
analysis from the BAR Expression browser program (BAR Toufighi et al. 2005) differs from
the other database analysis. So, the graphical format of the co expression network is obtained
for the Figure 5 and 6. CORNET (De bodt et al. 2010) has the flexible tool such as coexpression tool, which gives the co-express pattern in the graphical format through the
cytoscape software. From the CORNET database (De bodt et al. 2010), the co-expression
network for abiotic stress set of root and seedling is created as shown in Figure 7. The
following list of genes was analyzed: At3g07450, At3g52130, At5g0230, At5g62080,
At2g18370, At3g53980, At3g18280, At2g48130, At2g48140, At3g22620, At1g05450,
At1g05450, At5g13900, At2g37870, At5g59320, At4g33550, At5g59310. In this network, the
correlation coefficient was set as greater than 0.8 and red colour symbolize node and the dark
blue colour is the maxium coexpression of genes. When the stress set network was analyzed,
the different biological processes in the lipid transport were observed. Data obtained from the
BiNGO 2.3 annotation plugin shows that lipid transport gene not only aids in transport but
interestingly it also aids other biosynthetic process in the cell as shown in the Figure 8. There
are 471 genes in the network, in which 53 genes were annotated with stress response while 73
genes annotated with response to stimuli Go term. But only 18 genes were annotated with
lipid transport GO term as shown in Table 1.
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Figure 6 Coexpress pattern of the Arabidopsis nsLTPs gene from the novel tool, Expression
angler (Bio Array Resource).
Figure 7 Graphic view of co-expression pattern for stress induced nsLTP genes
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Figure 8 Over-represented GO terms detected in the stress induced nsLTP gene
Table 1. GO term annotation of coexpress network of the Arabidopsis abiotic stress set
GO term
Lipid transport
Response to oxidative stress
Response to stress
Response to stimulus
Response to chemical stimulus
Defense response
Phenylpropanoid metabolic process
Lignan metabolic process
Lignan biosynthetic process
Transport
Establishment of localization
Response to biotic stimulus
Localization
Phenylpropanoid biosynthetic process
Secondary metabolic process
Amino acid derivative metabolic process
P-value Corr p-value
2.29E-12
1.06E-09
3.04E-11
4.78E-09
4.07E-11
4.78E-09
4.13E-11
4.78E-09
2.94E-09
2.72E-07
2.49E-06
1.92E-04
3.42E-05
2.26E-03
6.04E-05
3.11E-03
6.04E-05
3.11E-03
2.24E-04
1.04E-02
2.46E-04
1.04E-02
3.03E-04
1.10E-02
3.09E-04
1.10E-02
4.70E-04
1.55E-02
7.13E-04
2.20E-02
1.15E-03
3.33E-02
When examine of same set of genes in tissuesuch as flower, leaf, root, seed from the
CORNET, correlation pattern is similar to Bio Array resources and the network topology is
obtained through the cytoscape as show in the Figure 9. Total number of genes in the network
is 223 and the co-expression is indicated as connecting edges with the number of 3798.
Annotation form BINGO 2.3 generate biological over represented network that may show
significant biological process like fatty acid metabolic process, pollen development,
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gametophyte development in the Figure 10. Analysis of co expression provides the new
insight of biological function involve in the lipid transport. Oxidative stress may be involved
in the lipid transport process which infer from the GO term enrichment analysis form the
Table 1 and 2.
Figure 9 Coexpression network of up regulated genes in the tissue set of Flower (72)*, leaf
(212)*, root (258)*, seed (83)*. The network is obtain form the yFiles organic layout organic
algorithm in cytoscape. White square is node and dark blue color shows the strong
coexpression correlation connecting two nodes. *Number of experiment in the database.
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Figure 2 Over-represented biological process GO term detected in the tissue set network.
Table 2 GO term annotation of arabidopsis upregulated nsLtps from the tissue set of
flower, leaf, root and seed.
GO term
Lipid transport
Transport
Establishment of localization
Localization
Lipid metabolic process
Developmental process
Sexual reproduction
Pollen development
Response to oxidative stress
Gametophyte development
Fatty acid metabolic process
Multicellular organismal development
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P-value Corr p-value
3.09E-21
7.69E-19
6.90E-07
5.89E-05
7.55E-07
5.89E-05
9.46E-07
5.89E-05
1.25E-04
6.21E-03
4.10E-04
1.70E-02
1.14E-03
3.94E-02
1.27E-03
3.94E-02
1.51E-03
4.17E-02
1.73E-03
4.30E-02
1.94E-03
4.36E-02
2.10E-03
4.36E-02
6 Discussion
LTPs were originally defined as the ability to transfer lipids between membranes in vitro.
However in the case of barley, carrot, Arabidopsis LTPs were observed outside the plasma
membrane in the cell wall (Sterk et al. 1991). This approach helps to raise the question of
what kind of biological role LTPs play.
6.1 Anatomical tissue-Genevestigator:
Initially, expression pattern of type G and other putative nsLtps gene are observed from the
Genevestigator meta-profile analysis tools (Zimmermann et al. 2004). Figure 1 and 2 shows
that the expression level in the heap map format, hence the maximum expressions were
observed in seed based on the mean values. These result is coincide with the previous studies
shown by Thoma et al. (1994) that the LTP genes were highly expressed in early development
in embryo cotyledons of A.thaliana. Few genes were highly expressed in the inflorescences
and roots. This indicates that a various plants also exhibit very high expression of nsLTP
genes in inflorescences (Fleming et al. 1992, Fleming et al. 1994, Thoma et al. 1994).
Expression according to anatomical tissue studies from both type G and other nsLTP genes
show that high number of nsLTP genes presence in the inflorescence, root and seed.
6.2 Abiotic stress-AtGenExpress:
AtGenExpress consortium provide the high quality, reliability and comparability of the
abiotic stress data set to facilitate in depth analyze of the stress response in Arabidopsis
(Kilian et al. 2007). Some Arabidopsis nsLTP genes greatly induced in root and shoot by
Nacl, mannitol and UV light. A 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). A previous
study by Khader (1996) suggests that the induction is observed in the plant tissue, but they do
not exhibit a constitutive expression of the gene.
In this study, the induction of nsLTPs gene is observed in the roots and shoots system of the
Arabidopsis through AtGenExpress visualization tool.
6.3 Co-expression analysis:
The main approach used in this study to develop co-expression network for stress induced
nsLTP genes and present the system level understanding of gene module that facilitate the
different biological function to carry out particular biological process (Linyong et al. 2009).
Co-express of gene network pattern generated by visual/manual and graphical format in this
study may be showed the similar and multiple biological functions. Hence, the co-expression
network analysis may find the regulatory function and metabolic pathway of gene (Hirai et al.
2004). In both stress and tissue set network, the oxidative stress may involve in the biological
process of the lipid transport. The GO terms for these biological processes were not
significantly over-represented, but they might also play a role in the lipid transport. Some
network analysis in the gene co-response studied by Nikiforova et al. (2005) showed that
sulfur deficiency which caused an enhanced lateral root formation through auxin and calciumrelated signaling pathways.
.
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6.3 Conclusion
In conclusion, no significant biological process are not observed in the network, but some
biological process like oxidative stress, defense response, phenylpropanoid biosynthetic
pathway may involve in the lipid transport process. This evidence is supported by Mao et al.
(2009) in Arabidopsis co-express network suggested that the biological processes which are
not significant over represented in the photosynthesis module; they might play a role in the
photosynthesis. This study provides new insight into biological process for stress induced
nsLTP genes.
7 Acknowledgements
Many thanks to my supervisor Johah Edqvist for his enthusiasm and devotion to the project
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