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BIOEN WORKSHOP ON METABOLOMICS OF SUGARCANE

Dec 7th, 2009

 

PROGRAM

8:30 h Welcome Coffee

9:00 h Opening Session

9:15 h Metabolomics of sucrose accumulation in sugarcane (abstract)

Carlos A. Labate
Departamento de Genética-ESALQ/USP - Av. Pádua Dias 11, CP 83 - Piracicaba-SP - Brasil

9:35 h Research on Biomass at the Max-Planck-Institut for Molecular Plant Physiology (abstract) 

Lothar Willmitzer
Max-Planck-Institut für Molekulare Pflanzenphysiologie - Am Mühlenberg 1, D-14476 Potsdam-Golm - Germany

10:15 h A systems approach to cellulose synthesis (abstract)

Staffan Persson
Max-Planck-Institute for Molecular Plant Physiology - Am Mühlenberg 1, D-14476 Potsdam-Golm - Germany

10:55 h Metabolomics: adding a powerful tool for sugar cane chemical profile studies (abstract)

Vanderlan da Silva Bolzani
Instituto de Química, NuBBE, Universidade Estadual Paulista - Araraquara, SP-Brasil

11:25 h Coffee Break

11:45h Monolignol profiling of lignin as selection tool for higher lignocellulose’s ethanol production efficiency (abstract)

Marcelo Ehlers Loureiro
Centro de Ciências Biológicas e da Saúde – CCB
Departamento de Biologia Vegetal, Universidade Federal de Viçosa - Viçosa, MG - Brasil

12:25 h Metabolomics-oriented Bioinformatics at the MPI for Molecular Plant Research

Dirk Walther
Institute of Biochemistry and biology - University of Potsdam

13:05 h Lunch Break

14:05 h Identification and characterization of metabolic QTL in Arabidopsis thaliana (abstract)

Jan Lisec
Max-Planck-Institute for Molecular Plant Physiology - Am Mühlenberg 1, D-14476 Potsdam-Golm - Germany

14:45 h  Adaptation of the metabolism of Serbania virgata to the storage compounds of the seeds (abstract)

Marco Aurélio Silva Tiné
Seção de Fisiologia e Bioquímica de Plantas – DJB - Ecofisiologia e Bioprospecção de Espécies Nativas
Instituto de Botânica, Secretaria do Meio Ambiente – SP - Brasil

15:25 h Metabolite and transcript interactions in response to the environment in Arabidopsis thaliana (abstract)  

Camila Caldana
Max-Planck-Institute for Molecular Plant Physiology - Am Mühlenberg 1, D-14476 Potsdam-Golm - Germany

16:05 h Biochemical networks related to carbon accumulation in sugarcane (abstract) 

Marcos Buckeridge
Department of Botany, University of São Paulo - Center of Science and Technology of Bioethanol (CTBE)
Instituto Nacional de Ciência e Tecnologia do Bioetanol (INCT-Bioentanol)

16:45 h Final Discussion

17:15 h Closing



ABSTRACTS


Metabolomics of sucrose accumulation in sugarcane

Carlos A. Labate - Departamento de Genética-ESALQ/USP - Piracicaba-SP - Brasil

In the last decade we have seen an extraordinary development of high-throughput methods for identifying and quantifying DNA, mRNA, proteins and metabolites which changed our view of plant metabolism. Systems Biology became a possible dream by integrating the information from genes, proteins and metabolites to study the complexity of metabolic networks, flux control and metabolic regulation. Today, a more applied view of these new technologies on sugarcane breeding provides an opportunity to use metabolite fingerprinting and profiling to identify desirable traits in plants from different populations. Understanding the regulation of plant metabolic pathways and the interactions between genes, phenotype, and environment is fundamental to functional genomics of sugarcane.

 

Plant breeders have selected in the last decades new cultivars of sugarcane with several traits of interest, such as high concentration of sucrose and reducing sugars in the stem, drought tolerance and disease resistance and higher productivity. The selection of such traits is related to modifications in metabolic composition which are still very little understood. Plant metabolomics has proven to be a valuable tool in assessing genotypic and phenotypic diversity, in defining biochemical changes associated with developmental changes and organ differentiation during plant growth and defining gene function. Our goal in the BIOEN project of metabolomics and proteomics of sugarcane is to develop a Systems Biology approach integrating transcriptomics, proteomics and metabolomics data, linking genotypes to phenotypes, to identify the genes associated with sucrose content in sugarcane. Our project proposes to build a data bank of metabolites and proteins, from different tissues and organs of sugarcane, expressed at different stages of development. We also intend to develop new computational plataforms linking the information of transcriptomics available in the SUCEST-FUN data bank for the identification of genes involved in sucrose accumulation

Research on Biomass at the Max-Planck-Institut for Molecular Plant Physiology

Lothar Willmitzer - Max-Planck-Institut für Molekulare Pflanzenphysiologie - Germany

Plant growth and thus biomass represent a major research focus at the MPI for Molecular Plant Physiology. Different aspects are dealt with in different research groups starting from primary energetic reactions such as photosynthesis via biosynthesis of different major polymers and metabolite classes such as cell wall, starch or TCA cycle intermediates and further including the study of various abiotic stresses (low nutrient (N,P,S) availability, cold or light stress).

The institute further follows an integrative approach combining genetics and transgenics with a variety of omics technologies (transcriptomics, proteomics, enzyomics and metabolomics) and using a suite of data analysis and data integration tools geared towards a systems biology approach.

In addition to giving an overview of the institute´s activities, one of the core platform technologies, metabolomics will be described in some detail with respect to both its methods and applications in plant breeding and diagnostics.

 

 

A systems approach to cellulose synthesis

Staffan Persson - Max-Planck-Institute for Molecular Plant Physiology - Germany

 

Plant cell walls are complex and dynamic structures mainly composed of highly glycosylated proteins and polysaccharides, such as pectin, hemicellulose and cellulose. Cellulose is the world's most abundant biopolymer, a key morphological component of the plant cell, and is also of great importance for various industrial processes. However, very little is known about how plants are making cellulose and how the need for cellulose production is communicated to the cell. We have utilized the fact that genes that appear transcriptionally coordinated with the cellulose synthase genes are functionally linked to cellulose biosynthesis. To assess these, and other, relationships in more detail we constructed interactive genome-level co-expression networks for five different plant species. We have targeted genes that appear in close vicinity to the cellulose producing gene modules, and that are present in more than one of the network contexts. Mutant analyses revealed that several of these genes are connected to cell wall synthesis, and may facilitate such diverse functions as signal transducers, cytoskeletal components, and carbohydrate modifying enzymes.

 


Metabolomics: adding a powerful tool for sugar cane chemical profile studies

Vanderlan da Silva Bolzani - Instituto de Química, NuBBE, Universidade Estadual Paulista - Araraquara, SP-Brasil

 

Plant metabolomics provides one of the pillars for studying the relation between the composition of complex and variable mixtures of plant-derived of great economic values, as medicines, crops, nutraceuticals, agrochemical, and, cosmetics. Thus, it is a promising approach for identification of metabolites, including the determination of metabolic pathways and genic pools, related to plant species, and can be useful for sugarcane studies, aiming to improve the resistance and/or its reaction to adverse abiotic and biotic factors. Considering that few metabolomic studies have been carried out on sugarcane so far, especially in Brazil, the development of analytical methods for the study of wide range metabolic profile of both primary and secondary metabolites of several sugarcane cultivars is mandatory, and encompasses a major goal of our proposal in BIOEN-FAPESP Program. Such approach is expected to result in the creation of a metabolomic database of Saccharum officinarum. As additional goals we propose the analysis of the metabolic dynamics during plant development and during infection and development of selected pathogenesis, considering the difficulties associated with their early diagnosis. In addition, considering our research group expertise in bioprospection of novel bioactive compounds from plant biodiversity, we also propose to perform the search for novel antifungal agents aiming the control of sugarcane pathogens associated with "sugarcane rust" and "sugarcane smut" using bench-top and/or field bioassays.

 

 

Monolignol profiling of lignin as selection tool for higher lignocellulose's ethanol production efficiency

Marcelo Ehlers Loureiro - Centro de Ciências Biológicas e da Saúde - CCB - Departamento de Biologia Vegetal, Universidade Federal de Viçosa - Viçosa, MG - Brasil

 

The analytical techniques developed in the field of lignin chemistry for pulp and paper processes like Py-GC-MS could be powerful tools to gain knowledge about lignin transformation, at molecular using pyrolysis coupled with mass spectrometry (PY/GC/MS), Analytical pyrolysis has been widely applied to study polymers and natural macromolecules on the basis of the identification by GC/MS of the products of thermal degradation. It has proven useful for gaining information on the structure of lignocellulosic polymers including several plants, residual lignin in some commercial products or after some industrial treatments, as well to study the effect of fungal attacks on the chemical composition of this polimer. Its advantage in regarding to nitrobenzene oxidation coupled HPLC analysis, includes the power to determinate various lignin units offering high sensitivity, rapid analysis time and no need for sample pre-treatment. It has been shown that the functional groups in the aromatic rings of lignin remain generallyunaltered in the course of pyrolysis, even though the propyl sidechains may be partially modified. We have optimized a Py-GC-MS method specific for sugar cane that needs only around 100µg, allowing unequivocal molignol fragment ions identification and relative quantification for several components. This method seems feasible to be coupled to a laser dissection microscopy, in order to get more restricted spatial information about changes in lignin composition and function. The Py-GC-MS protocol developed could be performed in just 20 minutes for sugar cane, allowing confident identification and relative quantification of about 16 lignin components. Further improvements are under way, now using a GC-TOF. Since that Brucker QTOFII allow the coupling of a Py-GC, we would like in the next future to use this new describe lignin structure and not solely lignin composition.

 

 

The results above have shown that no significant correlation can be seen between S/G rations and lignin content and no so wide variations were found for these plants. Study of other sugar cane populations (280 segregating individuals), where allows us to found only 8% relative difference for lignin content, and for S/G ratio, of 21,74%, similar results found in Populus that allow increases in 25% in the acid hydrolysis efficiency of this softwood, that probably could higher in sugar cane bagasse ethanol production. Risk of negatives effect of changes in lignin composition, in contrast to lignin content, is very low, since several studies addressing diversity of natural lignin chemistry have revealed that the flexibility of the lignin polymerization process allows plants to tolerate substantial changes in their lignin composition and/or incorporate phenolics other than the three common monolignols into their lignin We have made an extensive comparison about the determination of lignin composition using dry organ powder, cell wall extractive, or lignin extracted by Klasson method in sugarcane. Additionally, we can further speed up the determination of S/G, using a ball milling machine for 96 deep wells using very small amount of tissue, and preparing a simplified cell wall extraction using all the time 96 well plates. This approach could achieve the yield of 300 samples a week. We believe that our new attempts with GC-Q-TOF, a larger number of unidentified phenilpropanoid metabolites will be added to this group of identified compounds, and the use of quick PCA analysis of the ion chromatograms will speed up the discrimination for genotypic differences in some cell wall constituents. Coupling of strategies such as using FTIR as a first discrimination screening and neural network analysis (McCann et al., 2007), , could bring us unprecedented power to cell wall phenotype discover, and so better help to breeders getting better biomass for biofuels in sugar cane. Silent cell wall phenotypes could then be discovered and further characterized, extending the capabilities to understand the structure, synthesis and disassembly of the cell wall of sugarcane and other feedstock grasses important for biomass energy. High throughput and new methods are considered DOE-USA as a key points in the scientific development needed to turn cellulosic ethanol an attractive economical investment.

 

 

Identification and characterization of metabolic QTL in Arabidopsis thaliana

Jan Lisec - Max-Planck-Institute for Molecular Plant Physiology - Germany

To elucidate the connection between primary metabolism and plant growth is the main focus of our research. A particular interesting biological phenomenon often related to growth is heterosis or hybrid vigor. Arabidopsis as a model organism is well suited to investigate both, growth and heterosis. Therefore, we conducted several metabolomics experiments using large Recombinant Inbred Line (RIL) and Introgression Line (IL) populations generated from the Arabidopsis accessions Col-0 and C24 to identify metabolic Quantitative Trait Loci (QTL).

 

Within the presentation I will introduce some key concepts of quantitative genetics (RIL and IL populations, QTL Analysis) and describe our technological approach (GC-MS). Further, I present and discuss some of the obtained results which were published recently.

Adaptation of the metabolism of Serbania virgata to the storage compounds of the seeds

Marco Aurélio Silva Tiné - Seção de Fisiologia e Bioquímica de Plantas - DJB - Ecofisiologia e Bioprospecção de Espécies Nativas - Instituto de Botânica, Secretaria do Meio Ambiente - SP -Brasil

 

The monosaccharide mannose is reported to cause DNA fragmentation, inhibition of photosynthesis and germination on many plants. Seeds of Sesbania virgata (Cav.) Pers., however, have a storage compound based on mannose, which exposes the embryo to a high concentration of mannose during storage mobilization. This seed is, therefore, a model to study the metabolic adaptation to the storage selected by the species. The cultivation of the embryo in carbohydrate solutions allows the manipulation of the growth conditions to study the responses to sugars (sugar sensing). The analysis of the growth parameters showed that although the different monosaccharides are metabolized by the embryo, they generate different responses: while glucose inhibits photosynthesis, mannose is used as a carbon source without prejudice to photosynthesis. As opposed to mannose, glucose inhibited the embryo growth, and induced the storage of starch. The analysis by GC-MS of the metabolites allowed the comparison of the metabolic profiles of the organs grown under different carbon sources. The multivariate analysis of these profiles showed that the main differences are in the concentration of citrate, sucrose, glucose and myo-inositol. In the highest concentration of glucose and sucrose, there was accumulation of gluconic acid, suggesting a saturation of the metabolism, which did not occur in the mannose treatment, demonstrating the great adaptation of the metabolism to this monosaccharide. The use of different storage polysaccharides, therefore, allows the plant to use two metabolic pathways in an independent way: the storage mobilization and the photosynthesis with little interference from each other.

 

 

Metabolite and transcript interactions in response to the environment in Arabidopsis thaliana

Camila Caldana - Max-Planck-Institute for Molecular Plant Physiology - Germany

 

As sessile organisms, plants are challenged by multiple and sometimes fast changing environmental conditions, including those caused by temperature, light/dark cycles and nutrients. However, the mode in which multiple signals are integrated in the system level to adjust and reprogram the development for survival remains unclear. In the present work we are investigating the response of plants towards two environmental parameters, i.e. light and temperature. We have integrated the analysis of transcript and metabolite profiling data using high density time courses of plants transferred to eight different environmental conditions composed of three different temperature and four different light intensity regimes. In my presentation, I will discuss some aspects of these dynamic environmental responses. Further, I will focus on the importance of TOR pathway as integrator of environmental cues and plant growth.

Biochemical networks related to carbon accumulation in sugarcane
Marcos Buckeridge1,2,3 & Amanda Pereira de Souza1,3 - (1)Department of Botany - University of São Paulo,
(2)Center of Science and Technology of Bioethanol (CTBE), (3)Instituto Nacional de Ciência e Tecnologia do Bioetanol (INCT-Bioentanol) - Brasil

Biofuels are produced on the basis of substances composed mainly of carbon and hydrogen atoms that are stored in plant tissues. In order to produce biofuels from biomass, plants have to perform photosynthesis, i.e. to fix CO2 into carbohydrates that are subsequently transformed in more complex sugars, such as oligosaccharides (sucrose) and polysaccharides (starch and cell walls).
The easiness of retrieving energy from the glycosidic linkages of these carbohydrates depends on several aspects. Sucrose has to be fermented by yeast and the process leads to ethanol. On the other hand, starch and cell wall polysaccharides need to be firstly hydrolyzed chemically or enzymatically so that free sugars a produced and subsequently fermented to produce ethanol. Plants spend proportionally more energy to produce sucrose, starch and cell walls respectively. Whereas sucrose is stored in the vacuoles of parenchymatic cells, requiring large amounts of water to be maintained soluble, starch is packed into granules and cell walls in composites that require much less water and space to be stored. The form in which sugars are stored are relevant for the totality of energy that can be stored in plant tissues and this strongly influences the productivity of the process.Thus, it is crucial to understand how plants manipulated carbon after it is assimilated through photosynthesis and is finally stored in one of the forms mentioned above.
Sucrose, starch or cell wall polysaccharides require the use of progressively more complex metabolic networks that are controlled by genetic programs activated by the external (CO2, light, temperature and water) and exogenous factors (plant hormones, sugars and aminoacids). The modulation of these network connections within the plant, as a result of
the combined effects of the environment, determines what is called the source-sink relationship, which is the mechanism by which plants allocate storage compounds in their different body parts. In the case of sugarcane, carbon is allocated in culms as sucrose. The reason to accumulate high proportions of sucrose in culms is related to a preparation of the plant for flowering. This is why harvesting has to be quickly performed, otherwise, the flowering plants will use the storage sucrose in order to produce flowers and seeds, which are structures that demand large amounts of carbon and energy to be formed.
One interesting feature of sugarcane, which is related to the fact that it stores so much sugar in culms, is that it hardly accumulates starch in its tissues, being a typical sucrose storer.This indicates that the starch synthesis module is probably bypassed by the cell wall and sucrose syntheses ones. When sugarcane plants were grown under elevated CO2, i.e. twice the actual CO2 concentrations, an increase in photosynthesis was observed (de Souza et al. 2008) which were related to the light harvesting gene expression and light processing. The elevated CO2 also accelerated growth, increased biomass and changed slightly the Sucrose-Starch-Wall-Network (SSWN) relationship whereas less starch was produced. Usually, more input of carbon leads an increase of starch, but in sugarcane this seems to be the opposite. This study opened the way to investigate the likely candidates genes that connect these networks. The most likely to influence directly are the genes that encode ADPglucose pyrophosphorilase (ADPGpp), UDP-glucose pyrophosphorilase (UDPGpp), which are the enzymes that connect to production of starch and to sucrose/cell wall syntheses pathways respectively. They can be thought as primary targets to study because these are the enzymes that coordinate the distribution of sugars towards different carbon allocation modes in sugarcane plant system. Of secondary importance are SPS, SPP, INV, involved in sucrose metabolism, starch synthase and cell wall synthases. Our discoveries suggest that, in sugarcane, the modulation of the SSWN might be a interesting form to control carbon allocation in different forms in sugarcane so that in the future one could use strategies to induce carbon allocation in different forms and use sucrose, starch or cell walls to produce bioenergy.