Functional genomics of wood degradation and biosynthesis

Alex S. Rajangam

Licentiate Thesis Stockholm, Sweden 2005

Functional genomics of wood degradation and biosynthesis

Alex S. Rajangam

SRoy

S

Licentiate Thesis

chool of Biotechnology al Institute of Technology tockholm, Sweden 2005

© Alex S. Rajangam School of Biotechnology Royal Institute of Technology Albanova University Center SE-106 91 Stockholm Sweden Printed at Universitersservice US AB Box 70014 Sweden ISBN: 91-7178-250-8

Alex S. Rajangam (2005). Functional genomics of wood degradation and biosynthesis. Licentiate thesis from the School of Biotechnology, Royal Institute of Technology, KTH, Albanova University Center, Stockholm, Sweden.

ISBN: 91-7178-250-8

Abstract:

Forest biotechnology is a fast emerging field of research. The application of

biotechnological tools will enhance the quality of the forest products. The resultant value

added and environmentally sustainable products are an absolute necessity in the future. The

study of wood biosynthesis and degradation will result in enormous knowledge resources,

which can be used for exploiting wood properties. This thesis addresses questions representing

both wood degradation and biosynthesis.

The wood degrading fungus Phanerochaete chrysosporium is expression profiled with

the microarray technology. The objective is to understand the expression pattern of the

extracellular carbohydrate active enzymes (CAZymes) secreted by the organism. The data

obtained increases our understanding of gene expression upon growth on cellulose.

Wood biosynthesis is studied with the model wood forming tree species, Populus. The

plentiful data resources from the expression profiling during wood formation in Populus are

used as the platform of this work. One of the wood specific genes, PttMAP20, previously with

an unknown function is studied in this thesis. The immunolocalisation of PttMAP20 with

specific antibodies is demonstrated. The putative microtubule-targeting domain of the protein

is demonstrated microscopically and by using a biochemical binding assay.

Key words: Populus, xylogenesis, secondary cell wall, cellulose, hemicellulose, microarrays,

transcript profiling, Phanerochaete, wood degradation, microtubule, unknown genes.

© Alex S. Rajangam

Sammanfattning: Skogsbioteknologi är ett snabbt växande forskningsområde. Tillämpning av

bioteknologiska verktyg kommer att höja kvaliten på skogsprodukter. Förädlade produkter med

mervärde och miljömässigt hållbara produkter är en absolut nödvändighet i framtiden. Studier

av träets biosyntes och nedbrytning kommer utmynna i en enorm kunskapstillgång, som kan

användas för att exploatera träets egenskaper. Denna avhandling behandlar frågor både

gällande träets nedbrytnig och biosyntes.

Den tränedbrytande svampen Phanerochaete chrysosporium är uttrycksprofilerad med

mikromatris. Målet är att förstå uttrycksmönstret för de extracellulära kolhydrataktiva

enzymerna (CAZymer) som sekreteras av organismen. Den erhållna informationen ökar vår

förståelse för genuttryck till följd av tillväxt på cellulosa.

Träbiosyntes studeras i trädmodellorganismen Populus. De rika

informationstillgångarna från uttrycksprofileringen av träbildning i Populus används som

plattform för det här arbetet. En av de träspecifika generna, PttMAP20, som tidigare hade en

okänd funktion studeras i den här avhandlingen. Immunolokalisering av PttMAP20 med

specifika antikroppar uppvisas. Den troliga mikrotubuli-sökande domänen av proteinet

uppvisas mikroskopiellt och genom att använde en biokemisk bindningsanalys.

Courtesy translation by Mr. Anders Winzell, KTH, Sweden.

List of Publications This thesis is based on a paper, a manuscript and unpublished data. The papers are referred in the text by Roman numerals.

I. Amber Vanden Wymelenberg, Grzegorz Sabat, Diego Martinez, Alex S. Rajangam, Tuula T. Teeri, Jill Gaskell, Philip J. Kersten and Dan Cullen, “The Phanerochaete chrysosporium secretome: Database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium”, Journal of Biotechnology, Volume 118, Issue 1, 2005, Pages 17-34.

II. Alex S. Rajangam*, Henrik Aspeborg*, Kristina Blomqvist, Stuart Denman, Emma Master, Sophia Hober, Kathleen Piens, Vincent Bulone, Björn Sundberg, Ewa Mellerowicz and Tuula T Teeri, “PttMAP20, a novel microtubule-associated protein is highly expressed during xylogenesis in Populus tremula x tremuloides”, (Manuscript submitted in Plant Physiology).

In addition, previously unpublished data is included.

* A.S.R. and H.A. contributed equally to this work and should be considered as joint first authors.

Table of contents

1. Introduction

1.1. Biotechnology of biomass utilisation 1 1.1. Plant cell wall: the substrate 2 1.2. Microbial degradation of wood 3

1.2.1. Overview of fungal degradation of wood 1.2.2. Mechanism of degradation by P. chrysosporium

1.2.2.1. Lignin degradation by oxido – reductases 1.2.2.2. Hydrolysis of cellulose and hemicellulose 1.2.2.3. Expression profiling upon cellulose induction in vitro 1.2.2.4. Potential applications of P. chrysosporium

1.4. Wood formation 10 1.4.1. Xylogenesis 1.4.2. Expression profiling of secondary cell wall in model planta 1.4.3. Expression profiling of secondary cell wall in model tree

1.5. Frontiers of wood biotechnology 16

2. Aim of the present investigation 19 3. Materials and methods 21

3.1. Expression profiling in P. chrysosporium 3.1.1. Boutique arrays fabrication and hybridisation 3.1.2. Data analysis and SAM analysis

4. Results and discussion 25 4.1. P. chrysosporium - Cellulose induction in cultures

4.1.1. Expression profiling of transcript upon cellulose induction (unpublished data)

4.1.2. Peptide identification by LC- MS/MS (Paper I) 4.1.3. Identification of putative cellulose-binding iron reductase (Cir1)

(Unpublished data) 4.2. Populus tremula x tremeloides; Post genomic study of PttMAP20

(Paper II- Manuscript)

5. Conclusions and future perspectives 33 6. Acknowledgement 35 7. References 37

1. Introduction

Forests cover about 3 870 million hectares, or 30 % of the earth's land area.

Forests comprise the most significant energy reserve and are exploited for wood

products such as wood fuel, timber and wood pulp, and non-wood products including

medicines and food in the tropic and subtropics. Other societal uses of forest include

support to agricultural systems, employment generation for the forest inhabitants,

provision of recreational opportunities and the protection of natural and cultural

heritage [Global Forest Resources Assessment 2000,FAO, UN]. Forests also play

essential roles in soil and water conservation, biological diversity conservation and

mitigation of climate change.

The economic importance of forests for timber and paper industries has

brought enormous potential and scope for wood research. The need for the effective

use of the forest reserve for paper and pulp and the demand for environmentally

friendly processes has driven biotechnology into this area of research. Although

chemical industries have had a bigger role in forest industries to date, biotechnology

is slowly gliding in with tailored and environmentally friendly applications.

1.1. Biotechnology of biomass utilisation

The paper and pulp industries have been the backbone of many successful

economies of the world. The world’s total production of paper and pulp is mainly

shared by USA (28 %); Canada (14%); China (10%); Sweden (6%) and Finland (6%)

[Global Forest Resources Assessment 2000, FAO, UN]. Research in wood

biotechnology is beginning to investigate the effective use, improved performance

and the modification of fibres to engineer materials with low environmental impact.

Different areas of biotechnology are used in wood research for the modification and

processing of wood fibres, which is done either before or after the harvest of the tree.

The post-harvest treatment generally includes enzyme applications with wood pulp,

while pre-harvest treatment uses genomic approaches to introduce desirable

phenotypic traits in the plant during wood formation.

Post-harvest treatments: Post harvest processing has been traditionally

accomplished by chemical and physical treatments. The serious environmental

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consequences of these treatments have encouraged alternative technologies.

Environmentally friendly biotechnological treatments that have been used in the

recent past include biopulping with microbes, limited hydrolysis with microbial

enzymes, and bleaching with xylanases. Recently improved knowledge of wood

metabolism has identified potential enzymes that can be used in future for adding new

properties on the fibre surface.

Pre-harvest treatments: In vivo engineering of wood has been slow owing to

the long generation time of trees. However, plant breeding and selection by genetists,

as well as gene manipulation for desired phenotypes by molecular biotechnologists,

could contribute better yielding trees (Séguin, 2001; Boerjan, 2003). The genetically

manipulated lignin down regulated plants will ease the delignification processes by

the paper making industries (Gilles Pilate, 2002; Baucher et al., 2003). There is also

significant interest to obtain tree species with regulated production of cellulose.

1.2. Plant cell wall: the substrate

In plants, the rigid cell wall or the extracellular matrix is synthesised from

within the cell and organised outside the cell membrane. The first formed cell wall is

called the primary cell wall. During cell differentiation, the primary cell wall expands

and then is modified with new components forming the secondary cell wall (Fig.1).

Differentiated xylem cells with a secondary cell wall are called the secondary xylem

or wood. Given that wood is the Earth’s major source of biomass, it is fundamentally

interesting to study its physio-bio-chemical properties.

Chemistry of cell wall: In general the secondary cell wall is composed of

cellulose, hemicellulose and lignin. In poplar, the primary xylem cell wall is

composed of pectin (47%), cellulose (22%), xylan (11%), xyloglucan (6%),

glucomannan (1%) and proteins 10%. After differentiation, the secondary xylem cell

has an increased amount of cellulose (43-48%), xylan (18-28%) and glucomannan

(5%). The pectin and the xyloglucan content are not changed. The content of newly

integrated lignin is high (up to19-21%) while it was completely absent in primary

xylem cell walls (Mellerowicz et al., 2001). Besides carbohydrates and lignin the cell

wall contains major classes of cell wall proteins such as hydroxyproline-rich

glycoproteins (also called extensins), arabinogalactan proteins, glycine-rich proteins,

and proline-rich proteins (Ye et al., 1991).

2

The components of the secondary xylem represent an important substrate for

wood degrading microbes in nature and for many industrial products in the global

market.

Figure1: Diagrammatic representation of wood section, secondary xylem, secondary cell wall,

cellulose microfibril and microtubule orientation. Adapted from Biology of Plants (sixth edition), Peter

H. Raven, Ray F. Evert and Susan E. Eichhorn, W.H. Freeman and Company Worth Publishers.

1.3. Microbial degradation of wood

Microbial degradation of wood is an important biological process helping

nature to complete the carbon cycle by channelling the carbon stored as carbohydrates

back to carbon and hydrogen (Fig. 2). The microbes that degrade wood include

bacteria and fungi. Microbial degradation is either parasitic or saprophytic.

Saprophytic degradation can be specific to certain cell wall components or can

involve degradation of all cell wall components. There are many wood degrading

fungi such as Trichoderma, Fusarium, Penicillium, Ceriporiopsis, Humicola,

Aspergillus, and Phanerochaete. Cellulolytic bacteria include Thermonospora,

Cellulomonas and Clostridium.

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1.3.1. Overview of fungal degradation of wood

Fungi are heterotrophic eukaryotes and one of their major roles in the

ecosystem is bioconversion, including wood degradation. The saprophytic wood

degrading fungi are either unicellular or filamentous during the vegetative part of their

life cycle. Filamentous fungi are predominant wood degraders and their filamentous

morphology helps them to creep over and grow into the wood structures. In general

wood-degrading fungi secrete the enzymes that degrade complex cell wall polymers

and consumes the resultant smaller polymers, sugars and other products. Based on the

extent of degradation of wood components, the fungal species are referred to as wood

rot fungi, white rot fungi, brown rot fungi, soft rot fungi, etc.

There are many fungal strains that are used industrially for various

applications. Trichoderma reesei is one such extensively used forerunner. Many

enzymes from T. reesei are used for industrial applications such as pulp and paper,

textile, food processing, laundry etc. The white rot fungus Ceriporiopsis

subvermispora, though not well studied, is an effective delignifier and is used for

biopulping (R.A. Blanchette, 1991; Akhtar et al., 1992; Wall et al., 1993). In recent

times, the white rote fungus P. chrysosporium, with its unique ability to degrade

lignin and many organic pollutants, has emerged as a potential candidate for future

application in hazardous waste remediation and other industrial applications such as

paper and textiles.

Figure 2: Global carbon cycle depicted with the amount of carbon (billion tons) channelled through

biological and non-biological environmental units (Post, 1990).

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1.3.2. Mechanism of wood degradation by Phanerochaete chrysosporium

P. chrysosporium is an extensively studied white rot Basidiomycete. It is the

first wood rotting basidiomycete with a fully sequenced genome, readying for the

post-genomic era. Similar to other white rot fungi, it is able to selectively degrade

lignin from the wood making the wood white after initial infection; the remaining

cellulose is degraded later. The mechanism of P. chrysosporium is sequential, leading

to complete degradation of all wood components (Eriksson, 1993). Initially the fungus

degrades lignin with oxido-reductases and laccases followed by the degradation of

hemicelluloses and cellulose with various hydrolytic enzymes (Martinez et al.,

2004a).

1.3.2.1. Lignin degradation by oxido – reductases

Lignin is an integral part of the wood structure and cannot be isolated without

breaking its structure. This makes it difficult to study its biosynthesis and degradation

mechanism. Lignification in the later phase of xylogenesis increases wood strength

and hydrophobicity. Lignin is the most exposed chemical component of wood and

therefore lignin degradation is the first event during lignocellulose degradation by P.

chrysosporium.

Oxidases: Hydrogen peroxide (H2O2) is needed for the activity of peroxidases

that are involved in oxidising lignin. Many different peroxidases are produced by P.

chrysosporium (Fig. 8). They are characterised based on the transcript abundance

during secretion and protein identification in extracellular secretion. Genome

sequence analysis has identified the following putative oxidases: six copper radical

oxidases encoded (cro1 through cro6), five FAD-dependent oxidases, a glyoxal

oxidase (glx) and 3 putative glyoxal oxidase (GLOX)-like members. The genome

shows 4 aryl alcohol oxidase (AAO)-like sequences, a pyranose oxidase-like sequence

and a glucose oxidase like sequence (Martinez et al., 2004a).

Glyoxal oxidase (GLOX) is a copper radical oxidase and it catalyses the

oxidization of aldehydes to carboxylic acids, coupled to the reduction of dioxygen to

hydrogen peroxide. The purified GLOX is activated by lignin peroxidase, suggesting

a possible extracellular regulatory circuit for the control of H2O2 production by

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glyoxal oxidase, and control of lignin peroxidase activity by H2O2 (Whittaker et al.,

1996). Substrates for purified GLOX include formaldehyde, acetaldehyde,

glycolaldehyde, glyoxal, glyoxylic acid, dihydroxyacetone, glyceradldehyde and

methylglyoxal (Kersten et al., 1995).

The pyranose 2-oxidase (POX) is a glucose-oxidising enzyme produced by P.

chrysosporium when grown on lignocellulose. POX oxidizes various aldopyranoses

and disaccharides to the corresponding 2-keto sugars concomitant with the reduction

of O2 to H2O2 (de Koker et al., 2004a). The possible substrates, includes D-glucose,

D-xylose, L-sorbrose, and D-glucono-1, 5-lactone(Artolozaga et al., 1997). The

precise role of POX is still undetermined.

CH2OH

R

R

CHO

R

R

O2

O

O

O

OHO

FAD oxidases Copper radical oxidases

H2O2

a b

c

OR

OCH3

O

OH

OCH3

OR

HO

Peroxidasesα

β

γ

Cation radicaland phenoxyl

radicals

O2

H2O

Many lowMW products

''Enzymatic Combustion''

Figure 3: Major extracellular oxidative enzymes produced by Phanerochaete chrysosporium.

H2O2 production is physiologically coupled to peroxidase production. Major oxidases are (a) FAD

Oxidases; benzyl alcohol derivatives (alcohol oxidase (R = H or OCH3)) and (b) Glyoxal and copper

radical oxidases (methyl glyoxal). Lignin (Example: β-O-4 linkage (R = H or ether linkage to

additional monomeric units)) is oxidised by peroxidase to metabolizable low molecular weight

products that are used by the fungi. (The mechanism and the scheme adapted from Teeri TT (Teeri,

2004)

Peroxidases: Peroxidases catalyse one-electron oxidation of a wide range of

organic and inorganic substrates (Fig. 8). The plant peroxidase super family contains

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two families of lignolytic peroxidases such as lignin peroxidases (LiP) and manganese

peroxidases (MnP), which are also found in P. chrysosporium (Welinder KG, 1992).

There are ten lip genes, five mnp genes and interestingly there is a peroxidase gene

model pc.91.32.1, which is unlinked to all peroxidases but shares residues common to

both mnp and lip genes (Martinez et al., 2004a).

Lignin peroxidases, like other peroxidases, have a ping-pong mechanism i.e.

the enzyme is oxidised by H2O2 to give an enzyme intermediate that sequentially

oxidise two aromatic centres before returning to its original state (Kersten, 1992).

Manganese peroxidase is a heme peroxidase that oxidises Mn2+ to Mn3+ using

H2O2. The activity of this enzyme is stimulated by simple organic acids that stabilize

the Mn3+ and allow the peroxidase to oxidize organic compounds including phenolic

lignin model compounds (Gold et al., 1984; Glenn and Gold, 1985; Glenn et al., 1986;

Brown et al., 1990)

1.3.2.2. Hydrolysis of cellulose and hemicellulose

T. reesei is a well-studied cellulolytic fungus which degrades cellulose by

synergetic action of endoglucanases (endo-1, 4-ß-glucanase), cellobiohydrolases (exo-

1, 4- ß-glucanase) and ß-glucosidases (Teeri et al., 1987; Tomme et al., 1988).

Endocellulases degrade less crystalline cellulose, exocellulases act from the ends of

cellulose chains progressively and ß-glucosidases act on disaccharides and

oligosaccharides forming glucose. Similar cellulolytic mechanisms are found in P.

chrysosporium (Fig. 4a) (Eriksson, 1993).

P. chrysosporium degrades all components of cellulose and hemicellulose and

the genome predicts 240 putative carbohydrate active enzymes including 166

glycoside hydrolases, 14 carbohydrate esterases and 57 glycosyl transferases. There

are 40 putative endoglucanases, 7 exocellobiohydrolases and 9 β-glucosidases

predicted from the genome. The glycosyl hydrolases clearly out number the glycosyl

transferases, directly reflecting the priority of the organism for degrading wood by

extracellular enzymes (Martinez et al., 2004a).

Gene models derived from the P. chrysosporium genome predict the presence

of a number of enzymes that degrade xylan, mannan, xyloglucan, polygalacturonan

and rhamnogalacturonan. Other polysaccharide degrading enzymes that degrade

glycogen, mutan, chitin and β-glucans are also produced (Fig. 4b) (Martinez et al.,

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2004a). Notably, the pectate lyases that are normally present in wood degrading fungi

are absent in P. chrysosporium.

In addition to converting cellobiose to cellobionolactone, cellobiose

dehydrogenase (CDH) may also help in cellulose degradation via Fenton chemistry

(Mansfield S.D 1997). Fenton reactions generate highly reactive hydroxyl radicals

from peroxide H2O2 and reduced iron Fe(II). CDH is capable of generating Fe(II) and

H2O2.

Figure 4: Schematic representation of degradation of cellulose (a) and hemicellulose (b) by

filamentous fungi (Aro et al., 2005)

1.3.2.3. Expression profiling upon cellulose induction in vitro

P. chrysosporium can be grown in laboratory conditions on defined glucose

media, has high temperature optimum for growth, conidiates profusely and produces

basidiospores under established conditions (J.Kersten, 1992). Systematic degradation

of cellulose and hemicellulose by P. chrysosporium after the complete degradation of

lignin shows that the fungus can sense the extracellular carbohydrate source.

Secretion of extracellular enzymes is an energy consuming process, and plant cell

wall degrading enzymes are regulated by the available carbon source. This regulation

leads to the synthesis of specific enzymes that can degrade plant polymers as an

energy and carbon source (Aro et al., 2005). The expression of all degraditive

enzymes at the same time is possible due to the complexity of the cell wall. For

8

instance, in the cellulolytic fungus T .reesei the same compounds may induce the

expression of both cellulases and hemicellulases (Margolles-Clark et al., 1996).

Glucose repression: Substrate induced gene expression can be effectively

overridden by gene repression by using an easily metabolizable carbon source like

glucose (Zacchi et al., 2000; Yoshida et al., 2005). When the fungus is grown on a

cell wall polymer or synthetic polysaccharide, the polymer is too large to enter the

cell and therefore most degraditive enzymes are secreted. Glucose repression

effectively overrides the induction of these genes.

Expression profiling: Substrate induced expression has been studied since the

1980’s by reverse transcription and real time PCR. Enzymes involved in cellulose

degradation i.e. the endocellulase (Wymelenberg et al., 2002), exocellulases and

cellobiose dehydrogenases have been studied in cellulose-induced cultures (Covert et

al., 1992a; Covert et al., 1992b; Vanden Wymelenberg et al., 1993; Vallim et al.,

1998). Various studies have addressed transcript abundance during the induction of

chemical stress (Kurihara et al., 2002), iron (Assmann et al., 2003), xenobiotic

(Doddapaneni and Yadav, 2004, 2005) and polycylic aromatic hydrocarbons

(Hammel et al., 1986). Furthermore, proteomic analyses using mass spectrometry

have identified proteins secreted upon growth of P. chrysosporium on oak substrate

(Abbas et al., 2005).

The availability of the fully sequenced genome and high-throughput

expression profiling techniques present new ways to understand the spectrum of

secreted enzymes from P. chrysosporium. Expression profiling with microarrays

based on oligonucleotides has been used for profiling P450 monooxygenases and

regulatory proteins involved in signal transduction pathways (Doddapaneni and

Yadav, 2005).

1.3.2.4. Potential applications of P. chrysosporium

In addition to lignocellulolytic enzymes, P.chrysosporium secretes many other

enzymes that degrade complex organic substances. The organism can degrade many

toxic substance such as pesticides, polyaromatic hydrocarbons, polychlorinated

biphenyls and other halogenated aromatics (including dioxins), tri-nitro toluene

(TNT) and other toxic pollutants such as cyanide, azide, carbon tetrachloride and

9

pentachlorophenol (Kullman and Matsumura, 1996; Hawari et al., 1999; Cameron et

al., 2000; Reddy and Gold, 2000).

The omnipotent degraditive ability of P. chrysosporium leads to the following

potential applications.

Biosensors: P. chrysosporium has high sensing ability toward compounds in

nature that are of environmental concern. The enzymes from the organism can be used

in biosensors. A biosensor based on cellobiose dehydrogenase has been shown to

effective for detection of catecholamines (Stoica et al., 2004), the hemoflavoenzyme

cellobiose dehydrogenase (CDH, EC 1.1.99.18) had been used in an amperometric

redox polymer-based biosensor (Hilden et al., 2001) and the binding properties of

lignin peroxidase (LiP) against a synthetic lignin (dehydrogenated polymerizate,

DHP) was used in a resonant mirror biosensor (Johjima et al., 1999).

Ecological Bioremediation: Excellent biodegradative ability of P.

chrysosporium on various ecologically toxic materials makes it a potential candidate

for bioremediation. It has been proven effective for the bioremediation of

pentachlorophenol (Reddy and Gold, 2000), DDT (Corona-Cruz et al., 1999), oil

contaminants in soil (Ding et al., 2002) as well as textile effluent (Kunz et al., 2001)

and wastewater biofilm treatments (Wu et al., 2005).

Industrial application: The effective ligninolytic property of this organism

could be exploited to improve industrial delignification processes so far being done by

chemical and limited biological means. P. chrysosporium has already demonstrated

potential in bio-kraft pulping of softwood chips (Keller et al., 2003; Wolfaardt et al.,

2004) and biodegradation of volatile organic compounds in industrial off-gas

emissions (Qi et al., 2002).

1.4. Wood formation

A biosynthetic product of an autotroph, wood is the natural substrate for

degraditive assimilation by heterotrophs. Degradative processes of wood can be

viewed as mirror image events of wood formation. Polymers are the midpoint and the

monomers farthest from the midpoint (Fig.5).

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Figure 5: Lesson from nature, science and its application of bioconversion of the wood polymer. CO2

and H2O are fixed as plant polymer (wood biosynthesis) is degraded back again to CO2 and H2O (wood

degradation).

Wood formation involves complex biological processes such as cell division,

cell expansion, secondary cell wall deposition, lignification and programmed cell

death (Mellerowicz et al., 2001). The wood forming process is referred as xylogenesis

and the complete events of xylogenesis take place during one growth season.

Physiology of the xylem cell is influenced by the availability of water during the

season of a year. Bigger xylem cells are produced during spring compared to autumn

and this gives rise to the formation of annual rings. In annual plants like Arabidopsis a

single cycle of xylogenesis takes place in their lifetime.

1.4.1. Xylogenesis

Xylogenesis is the differentiation process of the meristematic cambium into

specialised xylem. The direction of the secondary growth makes xylem tissue the

prevalent biomass while phloem tissue becomes bark. Secondary xylem is divided

into different zones based on the biological happenings and biochemical content in the

respective zone of development. The zones are defined as A for cambial cell division,

11

B for cell expansion, C for late primary cell wall formation, D for secondary cell wall

formation and E for programmed cell death (Hertzberg et al., 2001b).

Secondary cell wall biosynthesis is a complex process that involves

coordinated regulation of several diverse metabolic pathways (Brown et al., 2005).

The secondary cell wall has three layers, S1, S2 and S3, which are different from each

other with respect to the cellulose microfibril orientation (Fig 1). The S2 layer has an

acute angle (less than 90˚) relative to the long axis of the cell, and makes the bulk of

the secondary cell wall (Atalla et al., 1993). The bulk of economically important

biomass is produced in the secondary cell wall. Expression profiling of secondary cell

wall formation will shed light on the coordinated enzyme activities involved in this

process. Resulting knowledge is a boon to research in pre-harvest fiber modification.

In the following section, expression profiling of secondary cell wall synthesis,

excluding lignification, is discussed.

1.4.2. Expression profiling of secondary cell wall in model planta

Thale cress, Arabidopsis, is the most commonly used model for plant

molecular biology. A short life span, small genome (480 Mbp, 50 times smaller than

pine) and easy breeding are some of the advantages of the thale cress for molecular

biology studies. Secondary cell wall biosynthesis can be induced in Arabidopsis by

decapitating its inflorescence (Lev-Yadun, 1994). The plant, when grown in short day

conditions can produce secondary cell walls in the hypocotyl and its anatomy is

similar to the angiosperms wood (Chaffey et al., 2002) (Fig. 6). Therefore,

Arabidopsis can also be used to study some aspects of wood formation (Nieminen et

al., 2004).

Secondary cell wall biosynthesis: Cellulose is synthesized by a group of

glycosyl transferases in family 2. Immunolocalisation and immunoprecipitation

studies suggest that the cellulose synthase complex must contain at least three CesA

polypeptides to be able to synthesize a single unit of microfibril (Gardiner et al.,

2003a; Taylor et al., 2003). Gene expression profiling experiments have shown the

upregulation of three of the secondary cell wall specific cellulose synthase (CesA);

CESA4, CESA7 and CESA8 and their respective mutants are irx1, irx3 and irx5 have

been studied (Oh et al., 2003; Brown et al., 2005; Persson et al., 2005). Microtubules

are believed to control the orientation of cellulose microfibrils in xylem cells.

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Microtubules are a heteropolmers of α-tubulin and β-tubulin dimers and both

monomers are found to be upregulated in the xylem compared to bark (Oh et al.,

2003).

Figure 6: Comparison of Populus stem and Arabidopsis hypocotyl. Cross-sections of the Populus stem

(A) and Arabidopsis hypocotyl (B) stained with TBO. X – Xylem, CZ – Cambium and P – Phloem

regions (Scale bar equal (A) 450µm and (B) 160µm (Chaffey et al., 2002).

Structural proteins such as hydroxyproline-rich glycoproteins (HRGPs),

proline-rich proteins (PRPs) and glycine-rich proteins (GRPs) are highly expressed

during secondary cell wall formation (Persson et al., 2005) and may also be involved

in secondary cell wall biosynthesis (Cassab and Varner, 1987). .

1.4.3. Expression profiling of secondary cell wall synthesis in a model tree

Populus is the first woody plant with a fully sequenced genome. Its relatively

small genome size (450-550 Mbp), availability of a large number of molecular

genetics maps, easy genetic transformation, easily vegetative propagation and

frequently occurring hybridisation are the advantages of Populus over other woody

species for tree molecular biology (Taylor et al., 2003).

Swedish consortial efforts brought the first light on the expression patterns of

wood-forming genes of Populus, a milestone in the history of forest genomics (Sterky

et al., 1998; Hertzberg et al., 2001b; Hertzberg et al., 2001a). Expression profiling

across wood forming zones resulted in a database of genes ranked according to their

comparative transcript abundances. The transcript list started with a relatively small

number of targets of around 5,000 of ESTs, while the present database describes over

1,00,000 ESTs (Sterky et al., 1998; Sterky et al., 2004).

13

Figure 7: (a) Cross section of hybrid aspen stem stained with Toluidine blue. The developmental zones

in the xylem are: A, meristematic cells; B, early expansion; C, late expansion; D, early secondary wall

formation; E, programmed cell death. (b) Schematic representation of the chemical component in the

developmental zone in xylem. (Figure adapted from Hertzberg M, Aspeborg H, et al. (Hertzberg et al.,

2001b) )

More than 450 genes out of the 13500 EST in the microarray are upregulated

in zone D (Fig.7). In different experiments, the gene expression in the zone D is

compared with the average level of gene expression over all of the zones (A-E), or,

separately with the gene expression in zone C or zone E. There are as many as 200

genes upregulated in zone D and 70 of them have unknown functions (Aspeborg et

al., 2005).

Carbohydrate active enzymes (CAZymes): The most significant event during

secondary cell wall biosynthesis is increased cellulose biosynthesis. In Populus

tremula x tremuloides about 10 putative cellulose synthase genes (CesA) have been

identified so far. These genes are designated as PttCesA1, A2, A3-1, A3-2, A4, A5, A6,

A7, A8 and A9. Higher numbers of CesA genes recently identified in Populus

trichocarpa suggest that there are additional CesA genes in the hybrid aspen.

PttCesA1, A3-1 and A3-2 are suggested to be specific for secondary cell walls (Djerbi

et al., 2005). Sucrose synthase, which channels sucrose to UDP-glucose, which then

polymerises to cellulose, is expressed in synchrony with the secondary cell wall

specific CesA’s (Hertzberg et al., 2001b). KOR is a cellulase that is believed to be

14

involved in cellulose biosynthesis in Arabidopsis, and PttCel9A, a poplar orthologue

of KOR, is found to be upregulated during the secondary cell wall biosynthesis

(Master et al., 2004; Szyjanowicz et al., 2004). UDP-D-glucuronate synthase

catalyzes the rate limiting step of pectin and hemicellulose biosynthesis, and is also

found to be expressed during secondary cell wall formation. Expression profiling

experiments resulted in several putative enzyme involved in hemicellulose

biosynthesis including putative glycosyl transferases, glycosyl hydrolases, xylanases,

β-galactosidase and xyloglucan endotransglycosylase (Aspeborg et al., 2005).

Microtubule associated proteins: Many microtubule-associated proteins are

found to be upregulated during secondary cell biosynthesis in Arabidopsis (Gardiner

et al., 2003b). These proteins are needed for the nucleation of microtubules and

related functions such as vesicular transport by the motor protein kinesin (Fig. 8). In

Populus, microtubule related proteins that are upregulated in zone D include 2 actin

related proteins, 8 kinesin related proteins, 2 MAP65/ASE1, 5 beta tubulin, a

dynamin-like protein, a vesicle associated membrane family protein, 3 TOR1-Family

proteins and ARP2/3 regulatory protein subunit NAPP.

Figure 8: Cartoon showing the movement of kinesin protein on the microtubule.

Unknown genes: Among the 70 genes that are highly expressed in zone D

there are 44 genes of unknown function. For these “unknown” genes only very basic

prediction of their function is offered by possessing protein features such as coiled

coils, transmembrane domains, a signal sequence, p21-rho binding domains, ring

fingers, tpr-repeat motifs, arp2/3 complexes, or a GPI-anchor.

15

1.5. Frontiers of wood biotechnology

Various fields of science and engineering are combined within the frame of

wood biotechnology. Completion of genome-sequencing projects for tree and wood

modifying organisms has brought post-genomic research to the frontier of wood

biotechnology. In Sweden, 270 million SEK has been invested for forest

biotechnology research in the past 10 years. Swedish consortia of various research

groups across the country have pioneered the field of forest biotechnology in the

world (http://www.swetreegenomics.se/). Table1 shows the funding allotted for some

of the post genomics projects ongoing in USA in Forest Genomics.

Year

Title

Total award

(US$) 2002-2005 2002-2006 2002-2006 2002-2007 2002-2005 2002-2005 2002-2006 2002-2007

Genomics of Loblolly Pine Embryogenesis Functional genomics of Host Virus Interactions. Functional genomics of Phytophthora- Plant Interactions. Functional genomics of Hemicellulose Biosynthesis. Transcriptome Response to Environmental Conditions in Loblolly Pine Roots. Functional genomics analysis of fruit flavor and nutrition pathways. Functional Analyses of Plant Gene Expression. Functional genomics of root growth and root signalling under drought

$ 1,380,910 $ 3,363,177 $ 1,891,617 $ 4,945,077 $ 1,651,752 $ 1,159,280 $ 1,135,486 $ 4,549,050

Table 1: Functional Genomics Projects funded by NSF, USA in 2002 (http://www.nsf.gov/).

P. chrysosporium is the first member of basidiomycetes with a fully

sequenced genome (http://genome.jgi-psf.org/whiterot1/whiterot1.home.html/). The

correlation of the already well-studied microbiology and enzymology of this white rot

fungus and the sequence-based annotation contributed clearer picture of the wood

degradation.

The Swedish P. tremula x tremuloides EST database contains over 100K

genes that have been collected by a concerted effort from KTH in Stockholm and

SLU and Umeå University in Umeå (http://www.populus.db.umu.se/). P. tremula has

16

a fully sequenced genome (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html/),

which positions Poplar as an ideal model for wood formation in trees (Bradshaw HD,

2000; Taylor, 2002). Furthermore, its recently deciphered genome sequence will

enable previous observations in plant science, forestry and paper and pulp research, to

be profiled at the level of transcripts, proteins and metabolites.

The two organisms discussed in this thesis, Phanerochaete chrysosporium and

Populus tremula x Populus tremuloides, have fully sequenced genomes and many

corresponding EST libraries. P. chrysosporium with a genome of size 30Mb and P.

trichocarpa with a genome of 480Mb, were sequenced by the DOE Joint Genome

Institute in the USA, and led by collaborative partners. These organisms provide good

models for studies of both wood degradation and biosynthesis. Genomic resources are

de facto alpha and omega of the biotechnology and the wood biotechnology is not an

exemption.

17

18

2. Aims of the present investigation

The general aim of the research was to identify potential enzymes and proteins

to facilitate both pre and post harvest engineering of wood fibres.

Wood degradation: (Paper I and unpublished data)

• Expression profiling of Phanerochaete chrysosporium in order to

understand the secretory profile of some carbohydrate active enzymes

(CAZymes) and unknown proteins upon cellulose induction.

Wood biosynthesis studies: (Paper II – Manuscript)

Characterisation of PttMAP20, a gene with previously unknown function by

• Cellular localisation studies with a specific antibody using confocal

microscopy and TEM.

• Development of a binding assay to demonstrate the binding of PttMAP20

to poplar microtubules.

19

20

3. Materials and methods

3.1. Boutique arrays fabrication and hybridisation

Boutique arrays with synthetic 35mers were designed to assess the feasibility

of transcript profiling representative genes potentially involved in cellulose

metabolism. The selected targets included 15 previously described sequences (i.e.

cDNAs in GenBank) and among these were the 6 closely related members of the cel7

family (Table 1). The known sequences also included gpd as a ‘housekeeping’ gene.

Nineteen targets lacking prior cDNA support were selected from the automated

genome annotation (Martinez et al., 2004b); (http://www.jgi.doe.gov/whiterot/).

These putative genes were predicted to encode extracellular proteins and were

structurally similar to proteins of known function including various glycosyl

hydrolases, oxidases, and an unusual cytochrome b-like reductase.

Preparation of the oligonucleotide microarrays: Oligonucleotide microarrays

were fabricated using oligonucleotides consisting of 35 nucleotides. Design criteria

were similar to those previously described (Kane et al., 2000; Rouillard et al., 2002)

and emphasized minimal secondary structure (no hairpins >3nt), similarity in melting

temperatures (Tm~80C), closeness to the 3’ end, low overall similarity (<75%) to

other coding sequences, and avoiding stretches >15nt of contiguous complementary

sequence. DNASTAR’s PrimerSelect (Madison, WI) was used to locate and

characterize oligonucleotides. Sequence similarity was checked against the entire

genome and against a separate database containing coding regions only. All design

criteria could not be achieved within the cel7 gene family, which is among the most

highly conserved families in the P. chrysosporium genome. In particular, the cel7A-

specific oligo is 83%, 77% and 80% identical to cel7A, cel7D, and cel7F,

respectively. The cel7E oligo is 80% identical to cel7F. None of these instances of

non-target similarity involved complementary stretches >15nt. All oligonucleotides

were synthesized with a C6 amino linker at the 5’ end to facilitate covalent attachment

to slide surfaces. The gene-specific oligos were arrayed on the Codelink activated

amine-binding slides (Codelink Bioarray system, Amersham Biosciences) with 24-

split pinhead Q Array robot (Genetix Ltd., UK). Each of the 34 genes was arrayed in

12 copies.

21

RNA extraction: P. chrysosporium RP78 RNA was extracted from glucose-

grown and cellulose-grown cultures using the RNeasy Maxi kit (Qiagen Inc,

Valencia, CA) according to the manufacturer’s instructions, with modifications.

Specifically, mycelia were harvested by filtration through Miracloth, snap frozen in

liquid N2, and ground to a fine powder with a pre-chilled mortar and pestle. Ground

mycelia were transferred to 50 ml conical tubes, and 15 mL RLC Buffer (RNeasy

Plant Mini Kit, Qiagen) were added. Tubes were shaken vigorously and the contents

forced several times through sterile syringes fitted with 18 gauge needles. The lysates

were centrifuged 5000 x g 10 min. in a swinging bucket rotor. One volume 70%

ethanol was added to the supernatants and shaken vigorously. The lysates were

loaded onto RNeasy maxi columns in 15 mL increments and spun 5-10 min. 5000 x g

until total sample volumes were loaded onto the columns. Fifteen mL RW1 Buffer

were added to the columns and spun 5 min. as above, followed by 10 mL RPE Buffer

and a 2 min. spin. An additional 10 mL RPE Buffer were added, and the columns

were centrifuged 10 min. 5000 x g to dry the membranes. To elute the RNA, the

columns were transferred to conical collection tubes, 0.8 mL RNase-free H2O were

loaded directly onto the membranes, incubated 1 min. room temperature, and

centrifuged 3 min. 5000 x g. The elution was repeated with an additional 0.8 mL

H2O. The eluates were quantified by optical density at 260 nm in a Hitachi U-1100

spectrophotometer. One-tenth volume of 3M sodium acetate and 2 volumes ethanol

were added, and the samples were stored at –200C until used.

Labelling: Probes were labeled with the CyScribe Post-Labelling Kit

(Amersham Biosciences). The method involved oligo (dT) primed reverse

transcription of the RNA with simultaneous incorporation of amino allyl modified

dUTP in the cDNA. After the synthesis, the modified cDNAs were purified by lysing

the RNA with NaOH and removing the free nucleotides and other reaction

components by using the GFX spin column (CyScribe GFX purification kit,

Amersham). The purified cDNA was then labeled with either the Cyst3 (550 nm) or

the Cyst5 (650 nm) dyes to allow differential hybridisation. The labeled cDNA was

dried by vacuum centrifugation for 1 hour and stored in the dark.

Hybridisation: Printed Codelink activated slides were incubated inside a NaCl

humidification chamber overnight (relative humidity of 75%). Before hybridisation,

22

slides were twice incubated in 0.1 M Tris, pH 9, 0.1% SDS at 50°C for 15 min. Slides

were then washed twice with deionised water, once in 4 x SSC (150mM sodium

chloride and 15mM sodium citrate (pH 7.0) with 0.1% SDS for 30 min, and twice

again with deionised water. Slides were centrifuged at 800rpm for 3 minutes to dry.

Hybridisation involved dissolving labeled cDNA in hybridisation buffer (5 x SSC,

0.1% SDS and 0.1mg/ml Salmon sperm DNA) at 95°c for 2 minutes, and then

applying the label to slides using 2.5ul per cm2 of cover slip. The hybridisation

chamber was incubated in a water bath at 50° C for 16 hrs. Following hybridisation,

slides were rinsed with 4 x SSC, and then washed successively in 2 x SSC, 0.1% SDS

for 5 min at 50° C, in 0.2 x SSC for 1 min at room temperature, and in 0.1 x SSC for 1

min at room temperature. Slides were scanned using an Agilent Microarrays scanner

G2565 BA (Agilent Technologies, Palo Alto, CA).

3.1.2. Data analysis and SAM analysis:

Target preparation: The following RNA samples were used for the

preparation of the targets: 1. total RNA from 3-day-old cultures grown on glucose as

the sole carbon source (3dG); 2. total RNA from 3-day-old cultures grown on Avicel

as the sole carbon source (3dA); 3. total RNA from 6-day-old cultures grown on

Avicel as the sole carbon source (6dA); and 4. a common reference mix with equal

quantities of the three RNA sources (Ref). The hybridisations were carried out using

labeled targets containing equal amounts of the Ref and 1. 3dG; 2. 3dA; or 3. 6dA

(Fig. 9).

Figure 9: The experimental design of microarrays hybridisation and image analysis.

Data analysis: Data from these hybridisations were analysed with GenePix

Pro 4.1 software (Axon Instruments, Foster City, CA). The hybridisation was repeated

23

4 times with the 3dG and 3dA probes, and twice for the 6dA probes. Hybridisations

resulted in a maximum of 48 values per gene (4 x 12 duplicates) for 3dG and 3dA

probes and a maximum of 24 values per gene (2 x 12 duplicates) for the 6dA probes.

The image analyses include manual removal of spots with signals, at or lower than the

background level. Owing to the low number of genes on the chip, the data set could

not be normalized by traditional methods. However, the relative significance of the

expression data obtained was evaluated by employing Significance Analysis of

Microarrays (SAM) (http://www-stat.stanford.edu~tibs/SAM/). The log 2 ratios were

used. The highest and the lowest values from the mean of the replicated hybridisations

for the 12 spotted replicates were removed before the analysis. Changes in expression

level were computed to compare 3dG vs. 3dA, 3dA vs. 6dA and 3dG vs. 6dA.

Scores (d) fold changes, and errors (q-value) were tabulated for genes with significant

positive or negative results.

All other procedures are described in paper I and II.

24

4. Results and discussion

4.1. Phanerochaete chrysosporium - Cellulose induction in cultures:

The experimental design of the comparison of the gene expression levels in

the cultures grown on different carbon sources is illustrated in Fig. 10.

Figure 10: Experimental set up of cellulose induction and microarrays experiment.

4.1.1. Expression profiling of transcript upon cellulose induction:

Data obtained by the comparative analysis (Fig 9, bottom panel) is presented

in Table 1. Of the 34 genes examined, 24 showed statistically significant changes of

expression under the different culture conditions tested. These genes can be roughly

grouped into three clusters.

The genes in the first cluster (genes 1-10, Table 2) were strongly induced

when grown 3 days on Avicel relative to when grown 3 days on glucose, but no

further increases in their expression levels were observed in prolonged cultivation on

Avicel (3dA vs. 6dA). As expected, this group includes genes encoding the major

exocellobiohydrolases as well as putative endoglucanases and hemicellulases.

25

T

e

T

a

No.

Gene Name

Putative enzyme or glycosyl hydrolase family

3G vs. 3A

3A vs. 6A

3G vs. 6A

1. cel7C Cellobiohyrdrolase type I

3.2

+

3.4

2. xyn11A Xylanase B 3.4

-

3.1

3. cel12A Putative Xyloglucanase / endoglucanase

3.0

+

3.4

4. cel7D Cellobiohyrdrolase type I

3.0

+

3.3

5. cel5B Putative Endoglucanase

3.0

-

2.7

6. cel7E Cellobiohyrdrolase type I

2.2

0

2.2

7. xyn10A Xylanase A 2.2

-0.6

1.6

8. cir1 Cellulose-binding iron reductase 2.2

-

+

9. cel6A Cellobiohyrdrolase type II

2.0

-0.5

1.4

10. cel5A Putative Endoglucanase

1.9

+

2.2

11. glx1 Glyoxyl oxidase -1.5

2.4

+

12. epg28A GH28 (Endo-polygalacturonase) +

1.9

2.4

13. gox1 Glucose oxidase +

1.6

2.5

14. cro2 Copper radical oxidase +

1.0

+

15. exp28B GH28 (Exo-polygalacturonase) +

1.0

+

16. aao1

Aryl-Alcohol oxidase

+

-0.3

+

17. aao3 Aryl-Alcohol oxidase

+

-0.4

+

18. cro6 Copper radical oxidase +

-0.4

+

19. cel12B Putative Xyloglucanase / endoglucanase

+

-0.6

-

20. cel61A GH61- endoglucanase

+

-0.6

-

21. cro3 Copper radical oxidase +

-0.6

-

22. pox1 Pyranose 2- oxidase +

-1.0

-

23. cdh1 Cellobiose dehydrogenase

+

+

+

24. gpdA Glyceraldehyde-3-phosphate dehydrogenase -

0

-

Scale -1 +1 +2 +3 +/-

able 2: Representation of the expression levels of the genes with statistically significant changes of

xpression. (SAM Analysis). Scale, (bottom line) Expression fold in Log2 ratio from the SAM analysis.

he cells in white indicate a positive or negative trend of expression but not statistically significant

ccording to the SAM analysis.

26

The putative cellulose-binding iron reductase (cir1, gene 8, Table 2) was up

regulated on cellulose (3dG vs. 3dA), which suggests a role for this enzyme in

cellulose degradation. The gene encoding glyoxal oxidase (glx1, gene 11, Table 2)

exhibited an interesting expression pattern with clear down-regulation upon the first 3

days of growth on cellulose followed by a strong induction upon prolonged growth on

cellulase (3dA vs. 6dA). This copper radical oxidase is believed to be physiologically

coupled to lignin peroxidases (Kersten and Kirk, 1987; Kersten, 1990), and prolonged

growth on Avicel is consistent with the idiophasic expression typical of the

ligninolytic system. Indeed, separate MS/MS analysis of 6 day Avicel cultures

detected lignin peroxidase isozyme H2 (Wymelenberg et al., 2005). A structurally

related copper radical oxidases, cro2, showed a pattern of expression similar to glx,

but transcripts of the additional cro genes were either not detected or down regulated

(cro3, cro6) after prolonged growth in Avicel

The genes in the second cluster (genes 12-15, Table 2) showed a weak trend

towards increased expression upon growth for 3 days on glucose instead of Avicel but

were clearly upregulated upon prolonged growth on Avicel (3dA vs. 6dA). This group

contains two putative polygalacturonases, a copper radical oxidase and FAD-

dependent glucose oxidase. Consistent with recent Northern blot analyses (de Koker

et al., 2004b), transcripts levels of another FAD oxidase, pyranose 2-oxidase (pox1),

was not up-regulated under these cultural conditions.

The third cluster (genes 16-22, Table 3) contains genes that show a weak trend

towards increased expression upon growth for 3 days on glucose instead of Avicel but

decreased level of expression upon prolonged growth on Avicel (3dA vs 6dA). The

gene encoding cellobiose dehydrogenase (cdh1, gene 23, Table 2) fell out of the

clusters described above with a slight positive trend of expression over the different

conditions, but no dramatic induction or repression of statistical significance.

4.1.2. Peptide identification by LC- MS/MS:

A database encompassing translations of the current 11722 gene models was

used to predict possible secreted enzymes. As a result, 268 putative secreted proteins

were identified using SignalP and TargetP algorithms. The secreted proteins were

then experimentally identified using mass spectrometry, resulting in 182 unique

peptide sequences representing 50 specific genes. Among these 50 genes, 24 genes

27

were among the secretome that were previously predicted by computational

algorithms. These 50 genes were classified as previously known proteins (18) (Paper

I; table 2), previously characterised glycosyl hydrolases (17) (Paper I; Table 2) and

uncharacterised genes (15) (Paper I; Table 4). The presence of the predicted secreted

proteins with low peptide scores was confirmed by RT PCR experiments (Paper I; Fig

2).

4.1.3. Identification of putative cellulose-binding iron reductase (Cir1):

Cir1, a putative cellulose binding iron reductase, was first discovered during

the transcript analysis and thereafter identified in the genome. The protein predicted

from the cDNA has a cellobiose dehydrogenase, cytochrome domain (CDH-

cytochrome) and a cellulose-binding domain (FCBD). The transcript abundance was

checked also with competitive RT PCR (Fig. 11). The transcript is completely absent

in the glucose grown cultures while it starts to appear with cellulose induction. The

experiment also confirmed the transcript expression pattern of Cir1 and Cdh1 was

similar to the microarrays experiments (Fig. 11 and Table 2).

.

Fig

10

Ci

ure 11: Quantitative RT PCR: Concentration of genomic DNA of Cir1 and Cdh1 from 1- 8; 10-10, -11, 10-12, 10-13, 10-14, 10-15 and 10-16 grams respectively and 9 – negative control. Expected size of

rl transcript 755bp, Cir1gDNA clone1082bp, Cdh1 transcript 846bp and Cdh1 gDNA clone 1102bp

28

4.2. Populus tremula x tremuloides; Post genomic study of PttMAP20 (Paper II-

Manuscript)

The data concerning PttMAP20 is described in Paper II and is therefore only

summarised here.

Gene discovery: PttMAP20 was first found in the expression profiling of wood

developing zone (Hertzberg et al., 2001b). PttMAP20, previously known as PttUnk1,

was found to have highest expression among the unknown genes that were relatively

upregulated in zone D (figure7a). The transcript presence was further checked in the

xylem sample using RT PCR. The expression level is relatively lower in zone C and

much lower in phloem, cambium, zone A and B (Fig. 7a).

Bioinformatics: PttMAP20 encodes a 177 amino acids long protein with a theoretical

molecular weight of 20.7 kD. The bioinformatic analysis revealed a putative

microtubule-targeting domain called the TPX2 domain. Expression of microtubule

related protein in addition to PTTMAP20 beta tubulin is also found to be highly

expressed in the zone D while the alpha tubulin has even expression across the wood

developing zones (figure7b).

Antibody production: Antibody was raised for the full length PttMAP20 gene

fragment cloned in pAff8c-3c vector and expressed in E.coli. PttMAP20 is immunised

both in a rabbit and a chicken. The rabbit Anti-PttMAP20 antibody was Affinity-

purified with the recombinant protein coupled to the NHS column and used for further

studies.

1. Cloning in pAff 8c 3c and expression in E.coli

2. Purification with IMAC column using HIS tag.

3. Immunization in rabbit and affinity purification with recombinant protein.

Figure 12: Antibody production scheme used in KTH Biotechnology, Sweden (Larsson et al., 2000;

Graslund et al., 2002).

Immunolocalisation of PttMAP20 and tubulin: The gene specific antibody was used

for the immunolocalisation of PttMAP20 in the wood section of Populus tremula x

29

tremuloides. The immunolocalisation was done also with the alpha tubulin antibodies

across the same regions to visualize the co-localisation of the microtubule and

PttMAP20.

Tissue level localization: The experiments showed the presence of the protein across

the wood forming section similar to the transcript abundance. The protein was

completely absent in the cambial region where there is active cell division. The

presences of the signals were prevalent in the cell types with secondary cell wall

thickenings while were depleting towards the zone D (Fig. 7a). The patterns of the

localization of microtubule were found to be present across all the zones.

Cellular level localization: The signals were found to be present closer to the cell

membrane, the border of living protoplasm. The signals of the protein were sporadic

clusters where as the microtubulin signals were uniformly present in the same region

of the cell. The PttMAP20 was found to be closer to cell membrane where

microtubule is present.

TEM studies: Further cellular localization is presently being done with transition

electron microscopy (TEM), using Anti-PttMAP20 labeled with gold particles. The

preliminary data shows the presence of the protein closer to the cell membrane and

also has a definite pattern. The proteins are present as patches on the cell membrane.

The labeling will be done with other membrane bound secondary cell wall related

proteins in future (Fig. 13).

A B

Figure13: Immunolocalisation of PttMAP20 in the ray cells showing the signals closer to the cell

membrane. B. Magnified white square of picture A showing the patched signals of PttMAP20 on the

cell membrane.

30

Microtubule binding assay: A binding assay was demonstrated to show the binding

property of TPX2 domain present in PttMAP20 protein. The experiment was done

with in vitro assembled Populus microtubule (Fig. 14).

A B

Figure 14: Invitro assemble of Populus microtubule. (A) Assembly condition disrupted by heating at

the start of the reaction (B) in vitro assembled Populus microtubule visualized with coomasie blue

staining.

The binding property of the PttMAP20 was demonstrated using the in vitro

assembled microtubule. The assay clearly showed the binding property of the protein

on the plant microtubule. This is also true with in vitro assembled bovine microtubule

(data not shown).

31

32

5. Conclusions and future perspectives The following information is the result of the research addressed in this thesis.

Wood degradation study: The secretory proteins of Phanerochaete chrysosporium

with cellulose induction were identified both at the level of transcript and protein

using RT PCR and mass spectrometry, respectively. Microarray expression profiling

for the selected carbohydrate active enzymes (CAZymes) was done with cellulose

grown cultures. A new gene called a cellulose-binding iron reductase (Cir1) was

identified during the study.

Wood biosynthesis study: The previously unknown protein PttMAP20 was

characterised. Bioinformatic analysis showed the presence of a putative microtubule

targeting domain in the PttMAP20 protein. The immunolocalisation studies showed

the presence of the protein closer to the cell membranes as clusters co-localising with

microtubule. A biochemical assay was performed to demonstrate the microtubule

binding property of the protein.

Future perspectives:

Wood degradation study: The use of bigger arrays (full genome array) will help

identify other proteins that are secreted during fungal growth on cellulose. There is a

drive to identify transcripts from P. chrysosporium grown on wood chips and

expression profiles during the wood degradation. The degraditive ability of the

organism on various organic pollutants could be exploited to use the organism as a

biosensor. For example, expression profile signatures of the organism grown under

defined conditions could be used to predict the composition of uncharacterized

environmental samples.

Wood biosynthesis study: The putative microtubule targeting property of PttMAP20,

has now been demonstrated at the gene and protein level. However, the physiological

function of the protein needs to be studied. Transgenic plants with RNAi to down

regulate levels of PttMAP20 are being done in collaboration with SweTree

Technologies, Sweden. The phenotype of the resulting plants will be helpful to

understand the function of the PttMAP20 protein in trees. Additional studies to

explore the relation of PttMAP20 to other genes that are involved in secondary cell

wall are underway. For example, co-localisation studies are being performed with

CesA components and other associated proteins. The structural determination and

binding property of the protein are of interest to determine the function of PttMAP20.

33

34

6. Acknowledgements Almighty, for blessing me with what I am.

Appa, Amma, Annangal, Akka, Annigal, Athan and all Kutties for all your

love, affection and prayers.

All the events, that brought me to Tuula for doing my Ph D. Thank you Tuula,

for giving me an opportunity to do my PhD studies with you. You have been my

friend, philosopher and guide during my research tenure in Sweden. Sweden was very

strange to me in terms of climate, culture and educational set-up and you stood beside

me during all my transitions.

All my teachers, especially from The American College you gave me the best

friends and teachers ever. Thank you Dr. Karunyal Samuel (AC, India), Dr. G.C.

Abraham (AC, India) and Prof. A. Ghanapathy (Biotech, BDU, India) for your

support during my research experiences. All my friends; Andrews, Leo, Sankar,

Sundar, Vimal, Kumar, Prasana, Revathy, Uma, Indumathy, 94BOTs, and 97PGBs all

you guys are in a way responsible for what I am now and you deserve a bit of curse

on that. All my friends in Sweden esp. Thoola family, Jaya, Doss family, Vasu family,

Kathleen family, Siju family, Noel, Pragash and Baskar thank you for keeping my

solitary moments occupied with laughter.

I would like to acknowledge my collaborators in various projects in a random

order for a fruitful collaboration; Dan Cullen, Ewa Mellerowicz, Henrik Aspeborg,

Harry Brummer III, Björn Sundberg, Sophia Hober, Peter Nilsson, Anna Ohlsson,

Totte Berglund, Anders Winzell, Carlotta Filling, Christian Brown and Christina

Divne. I would like to thank Annelie Waldén and Cecilia Laurell, Biotech, KTH for

their assistance in the printing of the microarrays and assistance to use SAM analysis

respectively. Kaj Kauko, KTH for his assistance to use fluorescent microscope. Stuart

Denman, Kristina Blomqvist, Emma Master, Kathleen Piens for being my

supervisors.

All my colleagues and friends at plan 2 for keeping a cordial and a friendly

working place. Alphonsa and Lotta for a nice administration.

35

36

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