Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius

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(1)UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Departamento de Biologia Vegetal. Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Fernando Vasco Rodrigues Cruz Pessoa. DOUTORAMENTO EM BIOLOGIA Biologia Molecular 2009.

(2) UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Departamento de Biologia Vegetal. Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Fernando Vasco Rodrigues Cruz Pessoa. Dissertação orientada pela Professora Doutora Maria Salomé Pais, Professora Catedrática da Faculdade de Ciências da Universidade de Lisboa. DOUTORAMENTO EM BIOLOGIA Biologia Molecular 2009.

(3) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. O presente trabalho foi financiado pela Fundação para a Ciência e Tecnologia, através da bolsa de Doutoramento SFRH / BD / 8020 / 2002.. A expertise adquirida ao longo da realização deste doutoramento contribuiu para a obtenção de resultados constantes das seguintes publicações:. Pessoa F, Sebastiana M, Acioli-Santos B, Kopka J, Pais MS The symbiotic interaction between Castanea sativa and Pisolithus tinctorius: a metabolic approach. (Em preparação). Figueiredo A, Fortes AM, Ferreira S, Sebastiana M, Choi YH, Sousa L, AcioliSantos B, Pessoa F, Verpoort R, Pais, MS (2008) Transcriptional and metabolic profiling of grape (Vitis vinifera L.) leaves unravel possible innate resistance against pathogenic fungi.. Journal of Experimental Botany. 59(12): 3371-81. Fortes AM, Santos F, Choi YH, Silva MS, Figueiredo A, Sousa L, Pessoa F, Santos BA, Sebastiana M, Palme K, Malhó R, Verpoorte R, Pais MS. (2008) Organogenic nodule development in hop (Humulus lupulus L.): transcript and metabolic responses. BMC Genomics, 9:445 Acioli-Santos B, Sebastiana M, Pessoa F, Sousa L, Figueiredo A, Fortes AM, Baldé A, Maia LC, Pais MS (2008) Fungal transcript pattern during the preinfection stage (12 h) of ectomycorrhiza formed between Pisolithus tinctorius and Castanea sativa roots, identified using cDNA microarrays. Current Microbiology. 57(6):620-5.. De acordo com o disposto no nº 2, art. 8, Dec.-Lei 388/70, o autor da dissertação declara que interveio na concepção e execução do trabalho experimental, na interpretação dos resultados e na redacção dos manuscritos enviados para publicação.. Fernando Vasco Rodrigues Cruz Pessoa. 1 / 108.

(4) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Acknowledgments To Professor Emeritus Maria Salomé Pais, who kindly accepted me to work on her lab, Unit of Molecular Biology and Plant Biotechnology, ICAT-FCUL, for all the encouragement during these years.. To Professor Joachim Kopka, for welcoming me in his lab thus allowing me to expand my technical and scientific knowledge.. To all my colleagues in ICAT: Aladje, Américo, Andreia, Bartolomeu, Cristina, Dora, Filipa, Hélia, Lena, Mané, Margarida, Mónica, Pedro, Silvia, Sofia, Susana, Vanessa and Violante for all their support, understanding and good working atmosphere.. A warmth thank you to Eva Sousa for always reminding me it was almost noon…. To all my friends and family for all the strength and support.. A very special thank you to my parents and my brother, for their friendship, support and help whenever I asked for it, and even when I didn’t.. And to my wife Rita, for always being there for me. Thank you for all your love.. Fernando Vasco Rodrigues Cruz Pessoa. 2 / 108.

(5) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Table of Contents Acknowledgments................................................................................. 2 Figure and Table Index .......................................................................... 5 Resumo.............................................................................................. 6 Abstract ............................................................................................. 8 Abbreviations .................................................................................... 10 1 – Introduction.................................................................................. 11 1.1. Mycorrhizae ........................................................................................11. 1.2. Ectomycorrhizae ..................................................................................14. 1.3. ECM Developmental Stages ......................................................................19. 1.4 The Castanea sativa x Pisolithus tinctorius interaction .........................................28 1.4.1 Castanea sativa Miller .................................................................................... 28 1.4.2 Pisolithus tinctorius (Pers.) Coker and Couch......................................................... 29 1.4.3 The ectomycorrhizal interaction Castanea sativa x Pisolithus tinctorius ......................... 29. 1.5 Metabolomics ...........................................................................................30 1.6 Aim of the Thesis .......................................................................................33. 2 - Materials and Methods ..................................................................... 34 2.1 Biological Material .....................................................................................34 2.2 Establishment of in vitro Interaction System .....................................................34 2.3 Sampling .................................................................................................35 2.4 Metabolite Extraction, Derivatization and Analysis ..............................................37 2.5 Generation of a Metabolite Response Matrix ......................................................39 2.6 Visualization Tools .....................................................................................41. 3 - Results and Discussion ..................................................................... 42 3.1 Metabolite Detection and Method Validation .....................................................43 3.2 Metabolite Profiling Analysis .........................................................................47 3.2.1 Saccharides................................................................................................. 49 3.2.2 Polyols ...................................................................................................... 55 3.2.3 Phosphates ................................................................................................. 60 3.2.4 Organic Acids .............................................................................................. 62 3.2.5 TCA Cycle Intermediates ................................................................................. 64 3.2.6 Amino Acids ................................................................................................ 66 3.2.7 Polyamines ................................................................................................. 71 3.2.8 Fatty Acids ................................................................................................. 72. 3.3 Integrated Analysis.....................................................................................74. Fernando Vasco Rodrigues Cruz Pessoa. 3 / 108.

(6) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 4 – Concluding Remarks ........................................................................ 81 References ....................................................................................... 83 Annexes ........................................................................................... 93 Annex A .......................................................................................................94 Annex B .......................................................................................................96 Annex C .......................................................................................................98 Annex D ..................................................................................................... 101 Annex E ..................................................................................................... 102 Annex F ..................................................................................................... 107. Fernando Vasco Rodrigues Cruz Pessoa. 4 / 108.

(7) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Figure and Table Index Figure 1.1. Mycorrhiza specific interaction structures.. 12. Figure 1.2. Proposed relationships between the distribution of biomes along environmental gradients and the roles of the prevailing mycorrhizal association in facilitating N and P capture by the functional groups of plant.. 13. Figure 1.3. Ectomycorrhizal structures.. 16. Figure 1.4. Simplified model of C metabolism in the ECM and AM symbiosis.. 18. Figure 1.5. Development stages of the ectomycorrhizal (ECM) interaction between Paxillus involutus and Betula pendula.. 20. Figure 1.6. Model of early signalling in symbiosis.. 24. Figure 1.7. The main expression patterns of plant and fungal symbiosis-regulated genes during the development of the ectomycorrhizal symbiosis.. 26. Figure 2.1. In vitro interaction system and control samples.. 36. Figure 2.2. The nine (9) time points under analysis.. 36. Figure 3.1. GC-MS chromatogram with the relative total ion count (TIC) of polar root metabolites.. 47. Figure 3.2. Accumulation patterns of selected Saccharides.. 52. Figure 3.3. Accumulation patterns of selected Polyols.. 57. Figure 3.4. Accumulation patterns of selected Phosphates.. 61. Figure 3.5. Accumulation patterns of selected Organic Acids.. 63. Figure 3.6. Accumulation patterns of selected TCA Cycle Intermediates.. 65. Figure 3.7. Accumulation patterns of selected Amino Acids.. 68. Figure 3.8. Accumulation patterns of selected Polyamines and Fatty Acids.. 73. Figure 3.9. Integrated metabolic map for the root interaction samples.. 75. Figure 3.10. Integrated metabolic map for the shoot interaction samples.. 76. Figure 3.11. Integrated metabolic map for the fungi interaction samples.. 77. Figure 3.12. Time course of the four stages characteristic of the ectomycorrhizal interaction and the main events occurring on each stage.. 78. Table 3.1. List of identified metabolites.. 44. Fernando Vasco Rodrigues Cruz Pessoa. 5 / 108.

(8) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Resumo As micorrizas são associações simbióticas resultantes da interacção entre fungos do solo e as raízes de plantas vasculares. A associação micorrízica origina um órgão especializado com morfologia própria – a micorriza – que é vital para o desenvolvimento de um sistema eficiente de captura de nutrientes. A formação de uma ectomicorriza origina uma série de interacções entre ambos os organismos e que podem ser agrupadas como segue: (1) contacto inicial entre as raízes e as hifas; (2) colonização da superfície da raiz com formação de um manto, penetração nas raízes e a formação da Rede de Hartig; e (3) órgão simbiótico funcional. A interacção ectomicorrízica entre Castanea sativa e Pisolithus tinctorius origina vantagens para as plantas micorrizadas tais como: aumento da tolerância a condições de stress biótico e abiótico e aumento da taxa de crescimento, do conteúdo proteico e da taxa fotossintética. De modo a investigar alterações metabólicas no decurso da interacção micorrízica entre Castanea sativa e Pisolithus tinctorius, foi efectuado um perfil metabólico por cromatografia gasosa acoplada a espectrometria de massa (GC-MS) em amostras de raízes, parte aérea da planta e fungo, quer em situação de controlo (ausência de contacto com o fungo) quer em situação de micorrização ao longo de 30 dias após contacto/interacção. A série temporal começa imediatamente antes da inoculação do fungo e prolonga-se pelas 6, 12 e 24 horas e 4, 7, 15, 20 e 30 dias de interacção. Foram analisadas um total de 51 condições e efectuadas 6 extracções independentes para cada condição, tendo-se obtido 306 perfis de metabolitos. Este enorme conjunto de dados, até agora não disponíveis, representou um imenso desafio e gerou uma enorme expectativa, quanto à informação decorrente da sua análise para a compreensão do metaboloma no processo de formação da ectomicorriza. A análise destes dados só foi possível graças ao desenvolvimento e adaptação de um método automatizado que permitiu a detecção e a quantificação relativa de um vasto leque de metabolitos: Açucares Simples, Polyols, Fosfatos, Ácidos. Fernando Vasco Rodrigues Cruz Pessoa. 6 / 108.

(9) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Orgânicos, Amino Ácidos, Polyaminas, fenilpropanoides e ácidos gordos. Estes perfis de metabolitos podem ser considerados como um “conjunto de imagens” que permitem desvendar as alterações que ocorrem, em simultâneo, em ambos os organismos em presença (planta e fungo micorrízico). Tendo em conta os resultados obtidos, bem como os mapas integrativos construídos, podemos concluir que os principais metabolitos implicados nas diferentes etapas de formação da micorriza são: (1) a nível da planta: amino ácidos relacionados com resposta ao stress e de defesa (prolina, alanina e glicina); metabolitos relacionados com a síntese e degradação da parede celular; sacarídeos (glucose, frutose e sacarose); acido galacturónico (um indicador de degradação da parede celular); amino ácidos aromáticos e metabolitos relacionados com a sinalização tais como myo-inositol, polyaminas, e galactinol; (2) a nível do fungo: a trehalose-fungo (marcador da presença do fungo); acido glucónico (promotor da formação de ectomicorrizas); glutamina (marcador de desenvolvimento do manto e da formação da rede de Hartig) e, tal como na planta, amino ácidos relacionados com resposta ao stress e de defesa (prolina, alanina e glicina) e metabolitos relacionados com a sinalização tais como myo-inositol, polyaminas, e galactinol. Este estudo pioneiro permitiu a obtenção de novos dados sobre esta importante temática (formação da estrutura micorrízica), ajudando a compreender alterações bioquímicas que ocorrem em cada um dos parceiros durante o estabelecimento da ectomicorriza entre Castanea sativa e Pisolithus tinctorius,. Palavras Chave: GC-MS,. Cromatografia Gasosa, Espectrometria de Massa,. Metabolómica, Perfil Metabólico, Micorriza, Castanea sativa, Pisolithus tinctorius.. Fernando Vasco Rodrigues Cruz Pessoa. 7 / 108.

(10) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Abstract Mycorrhizae are symbiotic associations resulting from the interaction of soil fungi and the roots of vascular plants. The mycorrhizal association originates a specialized organ with distinct morphology – the mycorrhiza – which is crucial for the development of an efficient nutrient uptake system. The establishment of an ectomycorrhiza encompasses a series of global changes in both organisms which may be grouped in three stages: (1) early hyphae–root contact; (2) root surface colonization with mantle formation; root penetration and subsequent Hartig net formation, and (3) mature symbiotic organ. The ectomycorrhizae interaction between Castanea sativa and Pisolithus tinctorius leads to several advantages to the mycorrhizal plants such as increased tolerance to biotic and abiotic stress conditions and also increased growth rate, protein content and photosynthesis rate. To investigate metabolic changes along the interaction between Castanea sativa and Pisolithus tinctorius, gas chromatography coupled to mass spectrometry (GC-MS) metabolite profiling was performed during the first 30 days of interaction in root, shoot and fungi samples, from both control and mycorrhiza (interaction samples). The time series included the time point immediately before inoculation, 6, 12 and 24 hours and 4, 7, 15, 20 and 30 days of interaction. A total of 51 different conditions were analysed, and considering that six independent extractions were performed for each condition, 306 metabolic profiles were obtained. This enormous data set represented a huge challenge, and its analysis has only been possible through the development and adaptation of automated methods that allowed the detection and relative quantification of a wide range of metabolites: Simple Sugars (Saccharides), Polyols, Phosphates, Organic Acids, Amino Acids, Polyamines, Phenylpropanoids and Fatty Acids. These metabolic profiles may be seen as a “set of pictures” which will help in understanding determine the changes that both organisms experience at each. Fernando Vasco Rodrigues Cruz Pessoa. 8 / 108.

(11) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. interaction stage and the possible signalling compounds used for the mutual recognition and communication between each one. Taking together our results and the integrated maps generated, we can conclude that the main metabolites crucial for the different steps of mycorrhizae formation are: at the plant level: stress and defence related amino acids (proline, alanine and glycine); cell wall synthesis and degradation related metabolites; saccharides (namely glucose, fructose and sucrose); galacturonic acid (an indicator of cell wall destruction); aromatic amino acids and signalling related metabolites such as myo-inositol, polyamines and galactinol; at the fungi level: trehalose-fungi (a marker of fungi presence); gluconic acid (a promoter of ectomycorrhizae formation); glutamine (a marker of mantle development and Hartig net formation) and, as at the plant level, stress and defence related amino acids (proline, alanine and glycine) and signalling related metabolites such as myo-inositol, polyamines and galactinol. This pioneering study will bring new insights on this important topic (ectomycorrhiza formation) by helping to understand the biochemical changes that each partner experiences during the establishment of the ectomycorrhizal symbiosis between Castanea sativa and Pisolithus tinctorius.. Key Words: GC-MS, Gas Chromatography,. Mass Spectrometry, Metabolomics, Metabolic. Profiling, Mycorrhiza, Castanea sativa, Pisolithus tinctorius.. Fernando Vasco Rodrigues Cruz Pessoa. 9 / 108.

(12) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Abbreviations 0-Ox.glut – Alpha-oxo-glutarate 2-Ala – Beta alanine Ac-CoA - Acetyl-CoA Aconit – Aconitic acid Asc – Ascorbic acid Cit – Citric acid DAG – Diacylglycerol DHAP – Dihydroxyacetone phosphate DNA – Deoxyribonucleic acid F1,6PP – Fructose-1,6-bisphosphate F6P – Fructose-6-phosphate Frc – Fructose Fumar – Fumaric acid G6P – Glucose-6-phosphate GA3P - Glyceraldehyde 3-phosphate GC-MS – Gas chromatography coupled to mass spectrometry GA3P – Glyceraldehyde-3-phosphate Glc - Glucose IP3 – Inositol 1,4,5-trisphosphate Isocit – Isocitric acid Mal – Malic acid myo-Ino – myo-Inositol Ox.Ac – Oxaloacetic acid P – Phosphate Group (PO43-) PAs – Polyamines PEP – Phosphoenolpyruvate Put – Putrescine RI – Retention Time Index RNA – Ribonucleic acid SAM – S-adenosylmethionine Spd – Spermidine Spm – Spermine Suc – Sucrose Succin – Succinic acid Amino Acids Ala – Alanine Arg - Arginine Arg.Succ - arginine-succinate Asp – Aspartic acid Asn - Asparagine Cit - Citruline Fernando Vasco Rodrigues Cruz Pessoa. Gln - Glutamine Glu – Glutamic acid Gly - Glycine Lys - Lysine Orn - Ornitine Phe - Phenylalanine Pro - Proline Ser - Serine Thr - Threonine Trp - Tryptophane Tyr - Tyrosine Val - Valine Nucleotides A – Adenine T – Thymine C – Cytosine G – Guanine U – Uracil Measurement Units atm - Atmosphere ºC – Celsius degree g – Gram g – Earth gravitic acceleration h – Hour N - Normal ng – Nanogram M – Molar mg - Milligram mJ – Millijoulle ml - Millilitre mM - Millimolar min – Minute p – Statistical expectation value pmol – Picomol rpm – Rotation per minute s – Second U – Unit ?g – Microgram ?l – Microliter ?m – Micrometer. 10 / 108.

(13) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 1 – Introduction 1.1 Mycorrhizae Mycorrhizae are symbiotic associations resulting from the interaction of soil fungi and the roots of vascular plants. The mycorrhizal association originates a specialized organ with distinct morphology – the mycorrhiza – which is crucial for the development of an efficient nutrient uptake system. In fact, mycorrhizae, and not roots, are the main organs of nutrient uptake by land plants and may have played a crucial role in facilitating the colonization of land by plants (Redecker et al., 2000; Smith and Read, 1997). Nutrient exchange between the partners is the key issue for the interaction. The fungus receives carbon from the plant, mainly in the form of carbohydrates, while the plant benefits from the fungus scavenging role of its widespread mycelium network for an increased water and nutrient uptake (Harley, 1953; Smith and Read, 1997). This is the most common and most important symbiosis in the world with nearly all plant families being able to form mycorrhizae with soil fungi belonging to all the main phyla, namely Glomeromycota, Ascomycota and Basidiomycota (Martin and Slater, 2007). There are different types of mycorrhizae. Their classification is based upon their phylogenies and characteristic structures of the specific interaction. Arbuscular mycorrhizae (AM) are endomycorrhizae and the most common and most frequent all over the world. About 80% of land plants form AM, including the majority of the plants used for agriculture purposes. AM symbiosis is formed by obligate symbiotic fungi from the Glomeromycota phylum. Hyphae enter the root tissues and develop inter- and intracellularly, forming specific structures: the intracellular arbuscules (Fig. 1.1 A;B). These are specialized structures responsible for the nutritional exchanges. In contrast to AM, a relatively small number of plants form ectomycorrhizae (ECM). ECM occurs mainly in temperate and boreal forests, where they are present in up to 95% of the short roots. ECM symbioses are formed by a large number of fungal species, mainly from the Fernando Vasco Rodrigues Cruz Pessoa. 11 / 108.

(14) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Basidiomycotina, but also from the Ascomycotina phyla. Fungi form a mycelial mantle around short lateral roots of their host and penetrate between the epidermal and cortical cells (the intercellular space), originating a highly branched structure, the Hartig net, which is the site for nutritional exchange between the fungal and plant cells (Fig. 1.1 C;D). Other mycorrhizal forms are the ericoid (ERM) and the orchid mycorrhizae, which occur specifically with plants from the order Ericales and the orchid family, respectively (Blasius et al., 1986; Kottke and Oberwinkler, 1987; Martin et al., 2001; Reinhardt, 2007, Smith and Read, 1997).. Figure 1.1 – Mycorrhiza specific interaction structures: arbuscular mycorrhiza (A and B) and ectomycorrhiza (C and D). A) Representation of the arbuscular mycorrhiza colonization process. After the formation of an intracellular coil in the first epidermal cell (the infection point), the fungus grows intercellularly to colonize the underlying cortex. It advances to the inner cortex cells where it forms the intracellular arbuscules (the site of nutritional exchange) (adapted from Reinhardt, 2007). B) Microscopic image of the characteristic intracellular arbuscules. Adapted from http://www.biology.ed.ac.uk/research/groups/jdeacon/mrhizas/ecbmycor.htm. C) Representation of the ectomycorrhiza colonization process. The fungus forms a mantle that surrounds the root. Some hyphae grow to the surrounding soil; other pass between the epidermal and cortical cells (always in the intercellular space) and form the Hartig net (the site of nutritional exchange). Adapted from http://www.backyardnature.net/f/mycorhza.htm. D) Microscopic image of the mantle and Hartig net.. Fernando Vasco Rodrigues Cruz Pessoa. 12 / 108.

(15) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Plants with ericoid (ERM), ecto- (ECM) and arbuscular (AM) mycorrhizae typically predominate under distinctive soil conditions (Fig. 1.2). In 1991 Read proposed that the respective fungal associates might, in each case, make contrasting types of contribution to plant nutrition. Since ERM and some ECM fungi have the potential to be directly involved in mobilization of N and P from the organic polymers in which they are sequestered, instead of just facilitating the capture of these nutrients in ionic form, at this time, research focus on moving away from reductionist approaches which consider only simple mineral ions as possible sources of the major elements, to give emphasis on the abilities of ERM, ECM and AM fungi to exploit N and P contained in substrates representative of those occurring in their distinctive natural environments (for a review see Read and Perez-Moreno, 2003).. Figure 1.2 – Proposed relationships, on a northern hemisphere based global scale, between the distribution of biomes along environmental gradients and the roles of the prevailing mycorrhizal association in facilitating N and P capture by the functional groups of plant (adapted from Read and Perez-Moreno, 2003).. Fernando Vasco Rodrigues Cruz Pessoa. 13 / 108.

(16) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Nutrient availability in Boreal and Temperate Forests favours the occurrence of ECM. Recent studies focusing on higher latitudes or altitudes (Tibbett et al., 1998; Tibbett et al., 1999; Schulze et al., 2000; Taylor et al., 2000; Lilleskov et al., 2002a,b) showed that both the biodiversity and the proteolytic capabilities of ECM fungal communities are greater in raw humus soils of northern boreal forests, where nitrification is undetectable, than in more southerly locations where mineral N enrichment occurs. The access to photosynthate from their autotrophic partners releases this component of the microbial biomass from carbon limitation, therefore providing it with the potential to play a major role in nutrient mobilisation, especially nitrogen (Read and Perez-Moreno, 2003). This explains why, although AM accounts for almost 80% of the total existing mycorrhiza, ECM are present in up to 95% of the short roots of plants of the Boreal and Temperate Forests and are essential players in these ecosystems.. 1.2 Ectomycorrhizae The ectomycorrhizal symbiosis has evolved repeatedly over the last 130-180 million years (LePage et al., 1997) and has had major consequences for the diversification of both the mycobionts and their hosts (Hibbett et al., 2000). The switch between saprotrophic and mycorrhizal lifestyles probably happened convergently and perhaps many times during the evolution of these fungal lineages (Hibbett et al., 2000). Ectomycorrhizal symbioses have a distinct host range allowing the formation of ECM on a limited set of trees and shrubs (Smith and Read, 1997). In temperate and boreal forests, where up to 95% of the short roots form ECM, the plants forming this type of mycorrhiza belong to the Pinaceae, Fagaceae, Myrtaceae and Betulaceae families (Wilcox, 1996) whereas the fungi partner belong to the Ascomycota and Basidiomycota phyla (Barker et al., 1998). In this symbiosis, both the plant and the fungi can associate simultaneously with many different counterparts. One plant may interact with different fungi in different locations of the root system, whereas the same fungus may interact with different plants, establishing links between them. Hence, the entire forest can be seen as a biological entity, connected in the. Fernando Vasco Rodrigues Cruz Pessoa. 14 / 108.

(17) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. underground by a “wood-wide web” of ectomycorrhizal fungi – a term coined by the journal Nature in order to highlight the work of Simard et al. (1997). This justifies why the ectomycorrhizal symbiosis plays an essential role in ecosystem stability. Besides the contribution in nutrient uptake, this ectomycorrhizal “wood-wide web” protects the root systems not only from the attack of pathogenic microorganisms but also from different stress conditions such as hydric stress and heavy metal soil pollution (Smith and Read, 1997). ECM has a huge beneficial impact on plant growth in natural (Read, 1991) and also in agroforestry ecosystems (Grove and Le Tacon, 1993). Structurally, ECM are characterized by the presence of a dense mass of fungal hyphae around the root. This fungal tissue, surrounding the lateral roots, forms the mantle (Fig. 1.3 B) which mediates the relationship between an outward fungal network of hyphae (the extramatricial hyphae or external fungal network) prospecting the soil and gathering nutrients (Fig. 1.3 A) and an inward labyrinthine and highly branched fungal network that forms the Hartig net (Fig. 1.3 C1 and C2) (Kottke and Oberwinkler, 1987). The Hartig net is the fungus-plant interface across which nutrients and carbon are transferred between the partners (Smith and Read, 1997). It results from the colonization of the epidermal and cortical cell layers of the root. ECM hyphae never form intracellular structures, as opposed to arbuscular mycorrhiza. The Hartig net is located in the intercellular spaces – the apoplast and the extracellular spaces – and is characterized by labyrinthine and highly branched fungal hyphae that increase the contact surface between the root and fungal cells (Tagu et al., 2002). The ECM mantle acts as a storage and buffer zone between the extramatricial hyphae and the Hartig net (Tagu et al., 2002).. Fernando Vasco Rodrigues Cruz Pessoa. 15 / 108.

(18) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Figure 1.3 - Ectomycorrhizal structures. A) A typical microcosm system (the external fungal network of hyphae) demonstrating the mycelium of Suillus variegatus growing from a Scots pine seedling (in Anderson and Cairney, 2007). B) Short roots ensheathed by an ectomycorrhizal fungus. The yellow mantle covers the root tip and rhizomorphs (i.e. hyphal bundles) are extending in the medium (in Martin et al., 2001) C1 and C2) Microscopic image of the ECM root structures. The Hartig net is pointed with arrows. We may observe the fungal mantle (M) surrounding the root and epidermis (E) and cortex (C) cells (adapted from: http://www.tau.ac.il/~ecology/virtau/safronov/mycorrhiza.htm).. The extramatricial hyphae are responsible for nutrient and water uptake. Increased efficiency of the fungal hyphae in nutrient and water absorption is a direct result of the increased area of absorption due to its small diameter (Rousseau et al., 1994). The release of several enzymes, namely proteases, phosphatases and oxidases, increases nutrient solubilization and consequently the absorption efficiency of the extramatricial hyphae (Bending and Read, 1995). Water and nutrients gathered by the hyphae are distributed to the remaining mycelium by a long distance transport system (Cole et al., 1998). Nitrogen and phosphorus uptake is greatly increased due to the mobilization of recalcitrant inorganic sources (Read, 1991; Perez-Moreno and Read, 2000) and,. Fernando Vasco Rodrigues Cruz Pessoa. 16 / 108.

(19) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. following the action of proteases, the assimilation of nitrogen from organic sources with the help of high affinity aminoacid transporters (Wallenda and Read, 1999). Glutamine, glutamate and asparagine are the main forms of nitrogen transferred to the host plant, while for phosphorus the ECM fungi accumulate polyphosphates as a reserve that may later on be transferred to the host plant (Smith and Read, 1997; Martins et al., 1999). In order to develop this extramatricial network, ECM fungi depend on the plant’s carbohydrates. The plant invests around 20% of its assimilates to feed the fungus (Barker et al., 1998). The extramatricial hyphae, the mantle and the Hartig net are active metabolic entities that provide essential nutrient resources (e.g. phosphate, nitrogen) to the host plant. The contribution of essential nutrients is reciprocal to the provision of a stable carbohydrate-rich niche in the roots for the fungal partner, making the relationship a mutualistic symbiosis (Martin et al., 2001). Sucrose, the major long-distance transport carbohydrate of most plants, is presumably exported into the apoplast of the symbiotic tissues and hydrolyzed by a plant-derived acid invertase, even though plants also have other enzymes capable of splitting the sucrose molecule – sucrose synthase (Lewis and Harley, 1965b; Salzer and Hager, 1991; Hampp and Schaeffer, 1999). The resulting monosaccharides are then taken up by the fungal partner (Lewis and Harley, 1965a; Palmer and Hacskaylo, 1970; Chen and Hampp, 1993). This assimilation by the fungus increases the “sink” effect of sucrose in the roots, leading to an increased production and transport of carbohydrates to the root system. Imported hexoses by the fungus are used for ATP generation, amino acid biosynthesis (carbon skeletons) and the formation of carbohydrate storage compounds (Martin et al., 1988, 1994; Hamp et al., 1995; Schaeffer et al., 1996; Kowallik et al., 1998). ECM fungi produce a series of fungus-specific sugars and sugar alcohols (Martin et al., 1985, 1987, 1988, 1998). Different pools of storage carbohydrates can be distinguished: oligosaccharides (trehalose), polyols (mannitol, arabitol and erythritol), and the long-chain carbohydrate glycogen. In addition to its function as a carbon reserve molecule, trehalose can also act as a stabilizer and protectant of proteins and membranes (Gadd et al.,. Fernando Vasco Rodrigues Cruz Pessoa. 17 / 108.

(20) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 1987) against heat (Bell et al., 1992), cold (Tibbett et al., 2002), and oxidative stress (Banaroudj et al., 2001). Its synthesis is mainly associated with the Hartig net hyphae (López et al., 2007). Figure 1.4 shows a simplified model of carbon (C) metabolism in ECM and also in AM symbiosis (Graham and Miller, 2005). Since AM symbiosis is far more studied than ECM, it is important to establish this parallelism in order to get more knowledge on the development of ECM symbioses.. Figure 1.4 - Simplified model of C metabolism in the ECM and AM symbiosis. Although sucrose is the primary transport carbohydrate in plants, ECM and AM fungi have no system for sucrose import. Sucrose can only be used as a C source by ECM and AM fungi provided it is hydrolyzed by cell wall bound invertases. Roots also possess sucrose synthase capable of splitting the sucrose molecule. What differentiates AM fungal C metabolism from ECM metabolism is its preference for importing glucose over fructose and the accumulation of lipids as the primary storage reserve (adapted from Graham and Miller, 2005).. Fernando Vasco Rodrigues Cruz Pessoa. 18 / 108.

(21) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 1.3 ECM Developmental Stages The establishment of an ECM symbiosis encompasses a series of morphological and developmental processes of both the colonizing mycelium and the plant lateral roots (Le Quéré et al., 2005). These processes depend on a finely tuned genetic programme operating on cell behaviour. A specific set of signalling molecules and growth factors promote cell divisions and tissue size, whereas proteins control the orientation of cell divisions, oriented cell rearrangements, and hence tissue shape. Such cellular differentiation is shaping the mycorrhizal symbiosis (Martin et al., 2007). Unfortunately, knowledge on the molecular triggers (signalling molecules, transcriptional factors and regulatory microRNAs) leading to such changes in gene expression is limited (Martin, 2008). Molecules that control the interactions between symbionts are involved in (1) tropism of hyphae for host tissues (rhizospheric signals); (2) attachment and invasion of host tissues by hyphae; (3) induction of organogenic programmes in both fungal and root cells (hormones and secondary signals); (4) facilitating mycobiont survival of the plant defence responses (fungal effectors); and (5) coordinating strategies for exchanging carbon and other metabolites for in planta colonization and growth of the soil fungal web vs soil gathered minerals (Martin et al., 2001, 2007). Global changes that occur during the establishment of the ECM symbiosis may be grouped in three stages: (1) early hyphae–root contact; (2) root surface colonization with mantle formation; root penetration and subsequent Hartig net formation, and (3) mature symbiotic organ. In figure 1.5 it can be seen the macro- and microscopic changes associated with each stage of development in the ectomycorrhizal (ECM) interaction between Paxillus involutus and Betula pendula. The early hyphae–root contact stage, which consists on the preinfection and adhesion, involves the first communication events and also the attachment and initial invasion of host tissues by the hyphae. Host plants release critical metabolites to the rhizosphere that are able to trigger fungus spore germination, growth of hyphae towards the root, and the early developmental steps of mycorrhiza formation. Fernando Vasco Rodrigues Cruz Pessoa. 19 / 108.

(22) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Figure 1.5 – Development stages of the ectomycorrhizal (ECM) interaction between Paxillus involutus and Betula pendula. Left panels: macroscopic views of the development of ECM root tips over time, starting at day 0 and ending at day 21. Birch seedlings were placed on a mycelium mat grown on a cellophane-covered agar plate. Arrows indicate the root apical region and lateral roots that developed into ECM root tips. Right panels: microscopic views showing cross sections of ECM root tips at different stages of development. At day 2, fungal hyphae have started to aggregate around the root tip, but this primary mantle remains loose and is easily separated from the root (arrow). At day 4, a high density of fungal hyphae is visible around the root tips as the mantle is forming. Two layers can be distinguished in the mantle, an inner layer consisting of apparently more closely adhered hyphae and an outer layer with more sparse hyphae (doubleheaded arrows). Note that the fungal hyphae have started to penetrate between the root epidermal cells (arrows). At day 8, the Hartig net has reached the anticlinal walls of the cortical root cells (arrows). At day 14, the Hartig net is fully established, i.e., the epidermal cells are fully surrounded by fungal hyphae (arrows). No major structural changes were observed at day 21, but a detectable labyrinthine structure produced from hyphal branching (arrow) indicates that the plant epithelial cells are extensively enveloped by fungal hyphae (adapted from LeQuéré et al., 2005).. Fernando Vasco Rodrigues Cruz Pessoa. 20 / 108.

(23) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. The continued hyphae invasion of plant roots triggers the second stage – the mantle and Hartig net development. As the mantle is being formed, the hyphae swell and branch isolating the epidermal layer of the root from its substract. The aggregating hyphae continue the colonization of the root epidermis which is followed by the inhibition and disappearance of pre-existing root hairs. From the inner zone of the mantle, the apoplast of the epidermal or cortical root cells is penetrated by hyphae, formimg the Hartig net. During formation of the Hartig net, the fungal cells proliferate between the root cells and the cell walls, and plasma membranes become highly invaginated. The host cells become multinucleated and rich in mitochondria and endoplasmatic reticulum, suggesting a high metabolic activity in these cells. At this level the exchange of carbon and nutrients between both partners may occur (Barker et al. 1998; Tagu et al. 2002). From the mantle, fungal hyphae extend outwards into the surrounding soil for translocation of nutrients back to the plant root. At this stage a functional mature symbiotic organ – the third stage – is established. The question is how these stages are regulated and what factors trigger their development. The pathways triggering the development of the ectomycorrhizal hyphal networks (mantle sheath, apoplastic Hartig net and soil fungal mats) involve the expression of genes that encode developmental proteins, enzymes and transporters that regulate the details of symbiosis development. These genes respond to rhizospheric and apoplastic signals released by the plant partner, positional information mediated by sensory molecules and nutritional cues (Martin et al., 2001). Recent studies, particularly those of Duplessis (et al., 2005) and of LeQuéré (et al., 2005), reported on dramatic alterations in gene expression taking place and required for symbiosis development. However, plant and fungal genetic switches that are necessary for ECM development remain unidentified to date (Martin et al., 2007). In AM symbiosis development, genetic switches have been identified. For a revision see Reinhardt (2007), AM symbiosis benefits from a close evolutionary relation with the root nodule symbiosis (RNS). This is the best-studied symbiotic interaction of plants, involving members of the legume family and bacteria, which are referred collectively as rhizobia (Stougaard, 2000). A striking. Fernando Vasco Rodrigues Cruz Pessoa. 21 / 108.

(24) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. parallelism may be found between these two interactions. In the RNS, flavonoids attract the rhizobia and initiate a symbiotic program that results in the transcriptional induction of several bacterial symbiosis genes, the Nod genes (Broughton et al., 2000; Stougaard, 2000). In the case of the AM symbiosis, the initial signals of the plant are the constitutively released strigolactones (Akiyama et al., 2005), which induce the hyphal branching and metabolic activity of AM fungi (Besserer et al., 2006). Plant exudates may attract not only symbionts but also saprotrophs or potential pathogens. Thus, a symbiosis signal allowing the recognition of a potential symbiont is of primary importance as the ‘door-opener’ in symbiotic interactions. In the RNS, flavonoid perception triggers the formation of the bacterial Nod factor, which signals back to inform the plant of the presence and identity of the rhizobia (Stougaard, 2000). In AM development, fungal hyphae that start to branch in the vicinity of a host root emit a first signal of unknown nature which activates the promoter of the symbiosis-related gene ENOD11 in roots (Kosuta et al., 2003). The establishment of a functional interaction in the RNS and the AM symbiosis involves a shared signalling pathway, here referred as the ‘common sym pathway’ (Parniske, 2004; Oldroyd and Downie, 2006). The first common component is a receptor kinase (SYMRK) (Endre et al., 2002; Stracke et al., 2002; Yoshida and Parniske, 2005), which is thought to integrate secondary signals from the perception of the Nod factor and putative Myc factors (Figure 1.6). A second component of the common sym pathway is a putative ion channel that is localized in the plastids (Ané et al., 2004; Imaizumi-Anraku et al., 2005), which might release a plastidial factor involved in signal transduction. Symbiotic signalling further requires a nucleoporin, a protein that has homology to a component of the nuclear pore complex (Kanamori, et al., 2006). Ultimately, symbiotic signalling triggers a nuclear calcium signal (i.e. calcium spiking), which is thought to be percepted by a calcium- and calmodulin-dependent protein kinase (CCaMK) (Levy et al., 2004; Mitra et al., 2004), which, in turn, induces the transcription of symbiosis genes. Besides the induction of gene expression, symbiotic signalling leads to specific changes in cellular organization and metabolism. How the events at the plasmalemma, plastidial, and nuclear levels are connected remains uncertain (black box in Figure 1.6). This ‘common sym pathway’ defines. Fernando Vasco Rodrigues Cruz Pessoa. 22 / 108.

(25) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. a partial overlap between the genetic programs for AM symbiosis and those for the nitrogen fixing RNS. The evolutionary implication is that the younger bacterial symbiosis has recruited perception functions from the ancient AM symbiosis (which has its origins more than 400 million years ago). It is tempting to speculate that the recent ectomycorrhizal symbioses (which first evolved around 180 million years ago) have also recruited the AM symbiosis SYM genes for signalling and development of symbiosis early steps (e.g. intraradicular accommodation) (Martin et al., 2007). Some of these genes (related to ion channels and Ca2+ signalling) have been found in the genome of Populus trichocarpa (Tuskan et al., 2006), a tree hosting both AM and ECM symbioses fungi. Although many pieces of the puzzle remain to be elucidated, the crosstalk among rhizospheric metabolites, the hormonal balance and signalling networks involving Ca2+ undoubtley play important roles in coordinating appropriate responses (Martin et al., 2007). Even though we may speculate regarding the mechanisms involved in the early signalling of ECM symbiosis, the transcriptional responses of fungal and root tissues progressing through the development of ectomycorrhiza have been reported by several authors. The works from Duplessis (et al., 2005) and LeQuéré (et al., 2005), on Eucalyptus–Pisolithus and Betula–Paxillus interactions using cDNA arrays allowed some insight on the main expression patterns of plant and. fungal symbiosis-regulated. genes during the. development. of. the. ectomycorrhizal symbiosis. In figure 1.7 a summary of the clusters of genes showing similar expression patterns identified by Duplessis (et al., 2005) and LeQuéré (et al., 2005), as well as the timeline of the interaction developmental stages is presented.. Fernando Vasco Rodrigues Cruz Pessoa. 23 / 108.

(26) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Figure 1.6 – Model of early signalling in symbiosis. Rhizobia produce nod factors (blue triangles), which are perceived by a receptor complex involving NFR1 and NFR5. An analogous pathway is predicted for the perception of arbuscular mycorrhiza fungi (AMF) derived hypothetical Myc factors (blue circles). Both signals are integrated by SYMRK and transduced via phosphorylation of an unknown substrate (X). Symbiotic signalling also requires a putative plastidic ion channel consisting of Castor (Ca) and Pollux (Po), which might release a plastidic factor (Y) that is required for signal transduction. Ultimately, a second messenger (Z) is translocated to the nucleus in a NUP133-dependent fashion (Nu), where it triggers calcium channels (purple) to release calcium (stars) from the nuclear envelope (i.e. calcium spiking). The calcium signal activates a calcium- and calmodulin-dependent protein kinase (CCaMK), which induces the transcription of symbiosis genes. Symbiotic signalling also leads to changes in cellular organization and metabolism. How the events at the plasmalemma, the plastid, and the nucleus are connected is unknown (black box). Some plant responses to AMF, involving transcription and the cytoskeleton, are independent of the common sym pathway (white arrows). Genetically defined signalling components are coloured in red (adapted from Reinhardt, 2007, adapted from Oldroyd et al., 2006). Expression pattern A contains transcripts identified as being involved in cell wall synthesis, stress and defence reactions presenting levels significantly higher in mycorrhizal roots at 4 days after inoculation declining thereafter as the symbiosis develops (figure 1.7). The highest expression takes place when fungal hyphae colonize the root cap and aggregate around root tips – the colonization stage. Examples of fungal transcripts included in this cluster are transcripts coding for hydrophobins hydPt-2 and hydPt-3, RGD-containing mannoprotein SRAP32, additional transcripts which are candidate markers for symbiosisrelated changes in cell wall. Plant transcripts include genes coding for. Fernando Vasco Rodrigues Cruz Pessoa. 24 / 108.

(27) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. metallothioneinlike protein, a hypersensitive-induced response protein, a pathogenesis-related (PR) protein, a cysteine proteinase inhibitor, a Bet v I allergen-related (major latex) protein, an elicitor-induced O-methyltransferase and a jasmonic acid-induced DNA-binding protein. The increased levels of PR proteins suggest that colonized root cells undergo defence reactions to restrict the fungal invasion. The RGD motif (Arg-Gly-Asp) can be found in proteins of the extracellular matrix. Integrins link the cytoskeleton of cells with the extracellular matrix by recognizing the RGD motif. RGD peptides interact with the integrin receptor sites, which can initiate cell-signalling processes making these transcripts molecular marker candidates for early symbiosis development (Tagu et al., 1996, 2001; Laurent et al., 1999; Duplessis et al., 2005). Expression pattern B encompasses early responsive genes presenting increased expression in mycorrhizal roots, attaining their maximum transcript levels at 7 days after contact – the mantle and Hartig net development stage (figure 1.7). This cluster contained several transcripts involved in hormone metabolism (i.e. ethylene-forming enzyme-like dioxygenase and an auxin induced aldo/keto reductase) and primary carbon metabolism (i.e. hexokinase, NAD-malate dehydrogenase, aspartate aminotransferase, isocitrate dehydrogenase, pyruvate kinase,. ATPase,. ATP/ADP. carrier. protein,. and. NADH. (ubiquinone). dehydrogenase) suggesting that carbon transfer between the host plant and the mycobionts is already taking place at 7 days and that this increased carbon flux stimulates glycolysis, the TCA cycle and respiration (Duplessis et al., 2005).. Fernando Vasco Rodrigues Cruz Pessoa. 25 / 108.

(28) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Figure 1.7 – The main expression patterns of plant and fungal symbiosis-regulated genes during the development of the ectomycorrhizal symbiosis. Transcriptional responses of fungal and root tissues progressing through the development of ectomycorrhiza were investigated in the Betula– Paxillus and Eucalyptus–Pisolithus interactions using cDNA arrays (Duplessis et al., 2005; LeQuéré et al., 2005). Groups of co-ordinately expressed genes were derived using hierarchical and nonhierarchical clustering algorithms. Major temporal patterns of induction or repression were observed with distinct groups of genes that have early, middle and late transcription responses to fungal colonization (adapted from Martin et al., 2007).. Fernando Vasco Rodrigues Cruz Pessoa. 26 / 108.

(29) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Expression pattern C encompasses genes showing maximal levels at 12 days after inoculation – Hartig net fully developed (figure 1.7). Several cellular functions are induced, including protein synthesis and fate (ubiquitin/ proteasome pathway, serine carboxypeptidase, translation initiation and elongation factors and ribosomal proteins), mitochondrial activity (citrate synthase, isocitrate dehydrogenase, ^-ketoglutarate dehydrogenase, malate dehydrogenase, ubiquinone-oxidoreductases, ATP synthase subunits), signaling pathway components (calcineurin B, protein kinase C inhibitor, ras, serine/ threonine protein kinase) and transcription factors. Transcripts involved in amino acid metabolism (NAD- and NADP-glutamate dehydrogenases, alanine aminotransferase,. high-affinity. methionine. transporter,. histidine. kinase,. homoserine kinase, nitrilase, _-1-pyrroline-5-carboxylate dehydrogenase) also showed their highest expression at this stage (Duplessis et al., 2005). Expression pattern D encompasses transcripts having an increased level during mycorrhiza formation and showing their highest expression at 21 days after inoculation (e.g. Outward Ammonium Transporter, 40S ribosomal protein S5) (Duplessis et al., 2005) (figure 1.7). Expression pattern E mainly contained genes whose transcript levels were significantly lower in symbiotic roots (figure 1.7). It encompasses genes coding for proteins such as the hydrophobin-related cerato-platanin, SnodProt1, metallothioneins, actin and actin-related proteins, proteins of the secretory system, translation elongation factor gamma, and hypothetical proteins likely involved in functions preferentially expressed in the free-living mycelium (Duplessis et al., 2005). There is still much to learn. In particular, it remains to understand exactly how the different host signals, which include strigolactones in AM symbiosis, integrate into a signalling network that alter the hyphal shape and metabolism, guide hyphae towards the root surface, and initiate and sustain in planta growth (Martin, 2008).. Fernando Vasco Rodrigues Cruz Pessoa. 27 / 108.

(30) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 1.4 The Castanea sativa x Pisolithus tinctorius interaction 1.4.1 Castanea sativa Miller European Chestnut, Castanea sativa Miller, also known as Sweet Chestnut, is classified in the order Fagales, family Fagaceae. It is a medium-sized to large deciduous tree attaining a height of 20-35 m with a trunk often 2 m in diameter. Its maximum height is reached after 60-70 years and the tree lives, in average, around 150 years. Chestnut trees are adapted to mild climates like the temperate forest regions and require adequate moisture for good growth and a good nut harvest. They thrive on neutral and acidic soils, such as soils derived from granite, sandstone, or schist, and do not grow well on alkaline soils such as chalk. It is presently distributed over the north of the Mediterranean, mainly in Portugal, Spain, Italy, Greece, Turkey and Croatia (Bergougnoux and Verlhac, 1978; Monteiro Alves, 1988). In Portugal, the European Chestnut is located mainly in the regions of Trás-os-Montes, Beiras and Minho and around Portalegre, ranging to an area of around 540 km2, according to the 2000 inventory (Martins, 2004). Sweet Chestnut is widely cultivated for its edible nuts that are used by confectioners and are also eaten roasted or boiled. In Portugal, it represents the most important exported fruit for countries like France (40%), Brazil (26%) and Spain (18%). Chestnut trees are also explored for its wood (Martins, 2004). Chestnut trees, like the majority of temperate forest trees, establish ectomycorrhizae (Alvarez, 1984). The mycorrhizal roots are superficial (5-20 cm below ground) and are concentrated around the trunk in a circle with a radius of 1-2 m. An inventory of macro fungus species present in chestnut orchards made in Trás-os-Montes (Portugal), revealed that 67% of the fungus were mycorrhizal, belonging to the Genus Amanita, Cantharellus, Lactarius, Tricholoma, Boletus, Hebeloma, Pisolithus, Paxilus and Russula (Martins, 2004).. Fernando Vasco Rodrigues Cruz Pessoa. 28 / 108.

(31) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 1.4.2 Pisolithus tinctorius (Pers.) Coker and Couch The fungus Pisolithus tinctorius (Pers.) Coker and Couch belongs to the phylum Basidiomycota, order Boletales, family Pisolithaceae. It is an ectomycorrhizal fungus with a wide geographic distribution (Cairney and Chambers, 1997). It is not very demanding regarding soil condition, being frequently found in locations with harsh environmental conditions like high temperatures and soils with high acidity, low water, low fertility and high levels of heavy metals (Martins, 2004). Pisolithus tinctorius has the ability to form ectomycorrhizas with a wide range of plant partners (more than 20 genus of host plants are known) and may be easily cultured in vitro. In the 1970’s, it was chosen for forestry programs and several plant inoculation protocols were established for this fungus (Cairney and Chambers, 1997). The easy availability of in vitro cultures allowed the study of its physiology and the establishment of the inoculation protocols with several different plant trees led to an increased knowledge of the Pisolithus tinctorius formed ectomycorrhizal ultra structure and ontogeny. The Pisolithus tinctorius x Eucalyptus globulus interaction has been the most used mycorrhizal interaction for the research of the molecular mechanisms underlying ectomycorrhiza formation, being considered as the model ectomycorrhizal symbiosis (Cairney and Chambers, 1997).. 1.4.3 The ectomycorrhizal interaction Castanea sativa x Pisolithus tinctorius Pisolithus tinctorius establishes an ectomycorrhizal interaction with the roots of Castanea sativa and the mycorrhizal chestnuts have an increased growth rate, protein content and photosynthesis rate (Martins et al., 1996, 1997, 1999). This particular interaction has been the focus of several studies. A hydroponic in vitro system has been developed for this particular interaction. Sebastiana (2006) and Santos (2006; et al., 2008), using a microarray approach were able to identify transcripts involved in the early steps of the interaction, both in the plant and in the fungal partner. This hydroponic system was also used by. Fernando Vasco Rodrigues Cruz Pessoa. 29 / 108.

(32) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Baptista (et al., 2007) to evaluate the involvement of reactive oxygen species during early stages of the interaction.. 1.5 Metabolomics A metabolite may be described as a compound which is internalized, chemically converted or secreted by an organism, but is not synthesized by DNA replication, transcription or translation. The origin of a metabolite is not exclusively dependent on the biosynthetic capacity or the genomic inventory of an organism. Metabolites may be exchanged between organisms, for example in plant microbe interactions (Kopka, 2006). They can also be viewed as the resulting end products of gene expression that will define the biochemical phenotype of a cell or tissue. The large scale analysis of metabolites is broadly called. metabolomics.. This. involves. the. quantitative. and. qualitative. measurement of large numbers of cellular metabolites thus providing a broad view of the biochemical status of an organism. Like the genome and the proteome refer to the complete set of genes and proteins, respectively, the complete set of metabolites present in a given organism is called the metabolome. (Bhalla et al., 2005; Sumner et al., 2003). Metabolomics has the ultimate goal of unbiased identification and quantitation of all the metabolites present in a certain biological sample (Fiehn and Weckwerth, 2003). However, the major limitation of metabolomics is its current inability to comprehensively identify and quantify all the metabolome. The genome and transcriptome consist of linear polymers of four nucleotides. The proteome is more complex, but is still based on a limited set of 22 primary amino acids. When surveying the metabolome, the chemical complexity is significantly greater. The chemical properties of metabolites range from ionic inorganic species to hydrophilic carbohydrates, hydrophobic lipids and complex natural products from the secondary metabolism. This makes it extremely challenging to analyze all of the metabolome simultaneously. Metabolic profiling refers to the detailed analysis restricted to the identification and quantification of a selected number of predefined metabolites in a given Fernando Vasco Rodrigues Cruz Pessoa. 30 / 108.

(33) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. sample. Despite escaping the opportunity of analysing all the metabolome, a comprehensive metabolic profiling analysis should cover multiple metabolic pathways in both primary and secondary metabolism. It should include carbohydrates, amino acids, organic acids, lipids/fatty acids, vitamins and other classes of natural products such as phenylpropanoids, terpenoids and alkaloids (Sumner et al., 2003; Halket et al., 2005). In metabolite profiling, de novo identification is mostly not a problem because all analytes under analysis have been selected based on previous identification and assumed biological relevance. Metabolite profiling is usually accompanied by rather focused biological questions and deep background knowledge, trying to cover all relevant compounds with authentic standards (Fiehn and Weckwerth, 2003). The selection of the most suitable technology must be a compromise between speed, selectivity and sensitivity (Sumner et al., 2003). Tools such as NMR are rapid and selective, but have relatively low sensitivity. Hyphenated mass spectrometry methods such as GC/MS and LC/MS offer good sensitivity and selectivity, but relatively longer analysis time. GC/MS is a relatively low cost alternative providing high separation efficiencies that can resolve complex biological mixtures. The requirement for the samples to be volatile is accomplished by derivatization. The term analyte may be used to address the chemical structure and compound which is submitted to GC/MS, detected and quantified. If the metabolite is not chemically derivatized, the analyte is identical to the metabolite. Also, a metabolite may have several analytes depending on the chosen derivatization reaction. The separation and analysis of complex mixtures may be performed with single quadrupole mass analysers. The utilization of an automated mass spectral deconvolution and identification system (AMDIS) enhances the ability to deconvolute and identify overlapping chromatographic peaks (Halket et al., 1999; Kopka, 2006). A major limitation of the metabolomics approach arises from the large biological variations (Sumner et al., 2003). Recent studies by Roessner and coworkers reported that the biological variability typically exceeds the technical variability (of GC/MS) by a factor of ten (Roessner et al., 2000). One way to reduce biological variance and strengthen the detected differences is to pool. Fernando Vasco Rodrigues Cruz Pessoa. 31 / 108.

(34) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. samples. By pooling multiple replicate plants the random individual differences (biological variability) will be reduced by statistical averaging and the focus will be on the shared variations due to the experimental conditions. A possible drawback of this pooling approach may be the undesirable dilution of specific up/down regulated metabolites (Sumner et al., 2003). Bearing this in mind, one must evaluate the problem in hand. When working with different individuals (for example, several plants germinated from different seeds), it may be convenient to reduce the random individual differences that are expected to occur. However, if the focus is on cultured cells, the individual random variability is already minimized and the pooling approach would not be necessary. All “omic” approaches rely upon bioinformatics for the storage, retrieval and analysis of large datasets; Metabolomics, even more. Statistical analysis must be performed to ensure good analytical rigor, but they require careful experimental design including replicate sampling, replicate analyses and the application of statistical tests. The development of means to visualize large amounts of data is also very important. Data visualization is the activity of displaying datasets in such a way as to allow direct visual identification of properties of the datasets. The use of reference biochemical databases such as KEGG (Kanehisa et al., 2004; Kanehisa et al., 2008), LIGAND (Goto et al., 2002) and EcoCyc (Karp et al., 2002) are important sources of information for metabolic networks attempting to identify the chain of causality that led to the observations. Whenever possible, metabolic data should be coupled with other “omics” technologies in order to provide an integrated picture encompassing all aspects of information flow from genome to metabolome and resulting phenotype (Sumner et al., 2003). Today, GC/MS is one of the most widely applied technology platforms in modern metabolomic studies. Examples range from post-genomic, high-throughput fingerprinting and metabolite profiling of genetically modified (e.g. Roessner et al., 2001a,b, 2002; Fernie et al., 2004) or experimentally challenged plant samples (e.g. Cook et al., 2004; Kaplan et al., 2004; Urbanczyk-Wochniak and Fernie, 2005). Some studies have focused on plant-microbe interactions, mainly in the model legume Lotus japonicus and the Root Nodule Symbiosis (RNS). Fernando Vasco Rodrigues Cruz Pessoa. 32 / 108.

(35) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. interaction with rhizobia sp. for nitrogen fixation (e.g. Colebatch et al., 2004; Desbrosses et al., 2005). Regarding mycorrhiza interactions, metabolic profiling studies covering multiple metabolic pathways in both primary and secondary metabolism are yet very scarce. The pioneering works from Lohse (et al., 2005), Schaarschmidt (et al., 2007) and Schliemann (et al., 2008) are important landmarks of metabolomic studies in arbuscular mycorrhizal interactions.. 1.6 Aim of the Thesis The main objective of this work was to investigate the metabolic changes occurring during the interaction between Castanea sativa and Pisolithus tinctorius using gas chromatography coupled to mass spectrometry (GC-MS). Starting with the first hours of contact and following up the developmental stages of the interaction, metabolic profiles of several key points were made. The time series included the time point immediately before inoculation, 6, 12 and 24 hours of interaction and 4, 7, 15, 20 and 30 days of interaction. These metabolic profiles may be seen as a “set of pictures” which will help us determine the changes that both organisms experience in each developmental stage and the possible signalling compounds used for the mutual recognition and communication between each other. For the success of the main objective, several previous goals had to be achieved. Metabolite detection and quantification in these two organisms was the first. Afterwards, a high throughput automatic method had to be developed and validated. This involved the manual selection and optimization of the detection window for each targeted metabolite. The last task was the analysis of the huge amount of data produced. This involved normalization, statistical treatment and development of a suitable data visualization technique to better convey all the information. This pioneering study will bring new insights on this important topic by helping to describe the biochemical changes that each partner experiences during the establishment of the ectomycorrhizal symbiosis.. Fernando Vasco Rodrigues Cruz Pessoa. 33 / 108.

(36) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. 2 - Materials and Methods 2.1 Biological Material Pure cultures of the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch (isolate 289/Marx), USA, were kept in petri dishes in the dark, at 25ºC, on solid Melin-Norkans (MMN) medium (Marx, 1969). A liquid culture was prepared by transferring pieces of fungal mycelium to culture flasks containing 200 ml of modified MMN medium (10 g/l glucose; phosphate and nitrate concentration were halved; malt extract, casein hydrolysate and agar were not added). Liquid cultures were kept in the dark, at 25ºC, without agitation. Seeds of Castanea sativa Mill. (obtained from “Centro Nacional de Sementes Florestais” – Amarante, Portugal) were sterilized with sodium hypochloride (2,5%) for 2 hours. To maximize sterilization, the pericarp (external skin) and the endocarp (tegument or internal skin) were removed (by careful peeling) and peeled seeds were again sterilized with sodium hypochloride (1,5%) and rinsed several times with sterile water.. 2.2 Establishment of in vitro Interaction System Sterilized seeds of Castanea sativa were suspended on a plastic net, inside sterile glass flasks, with 500 ml of sterile tap water. The plastic net, a germination platform, was placed just a few centimeters above the water (±2 cm) in order to avoid flooding the seeds and at the same time providing enough moisture. Germination was carried out at 25ºC, in the dark, until the plant shoot became visible. Afterwards, plants were transferred to a photoperiod of 16 hours of light (intensity of 35 µE.m-2.s-1) at 25ºC. After germination, the plants remained suspended on the germination platform with their roots inside the water – the interaction medium (figure 2.1).. Fernando Vasco Rodrigues Cruz Pessoa. 34 / 108.

(37) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. Fresh liquid cultures of P. tinctorius (1 week old) were filtered and the recovered mycelium was washed with sterile water and used to inoculate the C. sativa seedlings. Plants with a well developed root system were inoculated by transferring the washed mycelium to the interaction medium (the water). These will be referred as the interaction or “Myc” samples from now on. After gentle shaking, the flasks were maintained under the same growth conditions. Mock inoculations were done in glass flasks containing sterile tap water only (fungal control samples) and control plants were also kept without inoculation under the same growth conditions (plant control samples) – Figure 2.1. This in vitro interaction system has been previously described by Sebastiana (2006), Santos (2006) and Baptista et al. (2007).. 2.3 Sampling Root, fungi and shoot, from control and inoculated samples, were recovered at different time points up to 30 days after contact. At each time point (0, 6, 12 and 24 hours and 4, 7, 15, 20 and 30 days after plant fungus contact – figure 2.2), plants were randomly collected (inoculated plants and control samples) and frozen in liquid nitrogen. The frozen material was then ground in liquid nitrogen with a mortar and pestle and kept at -80ºC until analysis. For each time point, at least 8 independent biological replicate conditions were used (with two plants per replicate condition) and, after grinding the biological material, a pool was made with material from each independent replicate in order to smooth individual variations and focus on the global trends.. Fernando Vasco Rodrigues Cruz Pessoa. 35 / 108.

(38) BIOLOGY PhD Thesis, 2009 Metabolic profiling of the symbiotic interaction between Castanea sativa and Pisolithus tinctorius. A. B. C. Figure 2.1 – In vitro interaction system and control samples. A) Castanea sativa control plants; B) Castanea sativa x Pisolithus tinctorius interaction samples (“Mycorrhizal” samples); C) Pisolithus tinctorius control sample. Figure 2.2 – The nine (9) time points under analysis: 0 (starting conditions), 6, 12 and 24 hours and 4, 7, 15, 20 and 30 days after inoculation.. Fernando Vasco Rodrigues Cruz Pessoa. 36 / 108.