The International Consortium of Sugarcane Biotechnology sponsors a project in Dr. Andy Paterson´s lab at University of Georgia that tries to integrate genetic/QTL maps, ESTs and BAC libraries into a 'gene map' that will also serve as an initial physical platform of sequence tagged sites (STS) that can be used for sugarcane genomic sequence assembly. Most sequences mapped in the sugarcane database in BacMan belong to Arabidopsis, sorghum and sugarcane. Until this date (30/11/2004) almost 5,000 overgo sequences have been mapped anchoring 80,535 BAC sequences.
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A current major challenge is to explore the large amount of genomic information obtained by sequencing the EST collection generated by the Sugarcane EST Project (SUCEST) to develop new products (improved sugarcane cultivars) and to increase our current biological knowledge of a complex plant system. High throughput genome-wide transcriptional profiling methods are required to make use of all the information available. Due to reduced set-up costs and the availability of DNA analyzers, we chose to adopt the Serial Analysis of Gene Expression or SAGE approach. Serial analysis of gene expression (SAGE) has become an important tool for genome-wide gene expression studies, mainly in humans and mammals, but it is becoming more popular in the plant biology community. SAGE consists in isolating, sequencing and counting small tags (~10 bp), each representing individual transcripts, from a relevant biological condition (e.g. high x low yield; presence x absence of stress), yielding a qualitative and quantitative profile from the sampled transcriptome. Our group at the Plant Breeding Laboratory from CENA-USP has applied this approach to investigate and characterize genes differentially expressed under various contrasting conditions. The availability of a large EST collection turns SAGE an attractive approach, since tag annotation is specifically improved. Besides, the integration of SAGE data to macro- and/or micro-array information within the framework of the SUCEST-FUN Project will provide a more complete knowledge about sugarcane transcriptomics, improving chances of success to apply the information generated by functional studies.
Another field of interest of our group is to investigate sugarcane tolerance to heavy metals aiming to develop a strategy to adopt sugarcane in phytoremediation (remediation of sites contaminated with metals).
|1. Sugarcane Leaf Gene Expression Profile|
|Tercilio Calsa Junior
A transcriptional profile from field-grown sugarcane mature leaves of cultivar ‘SP80-3280' was obtained using SAGE standard protocols. Tags were extracted from .phd files (generated by Phred-Phrap after sequencing) using SAGE2000 and eSAGE combined with Microsoft Access. To date, 10,411 tags were isolated, comprising 5,660 unique sequences, from which 76.3% were detected just once. Annotation of tags was carried out by matching tags to NCBI/SAGEmap plant database (www.ncbi.nlm.nih.gov/SAGEmap), and to SUCEST CAP3-clusters database via BLASTN. The most abundant tags were annotated as representing genes for photosystem I and II subunits; ferredoxin and plastocyanin precursors; and a glycine-rich RNA-binding protein, with highest similarity scores to corresponding Zea, Oryza, and Hordeum and Arabidopsis genes. Photosystem and Z-scheme related tags were the most frequent, but also transcripts from CO2 fixation; stress responses; and primary/secondary metabolism were found. Noteworthy, several putative transcription factors were isolated.
|2. Sucrose Storing Internode Gene Expression Profiling|
|Tercilio Calsa Junior, Danielle Scotton
To identify genes differentially expressed in genotypes contrasting for sucrose content in stem internodes parenchyma, SAGE has been applied to establish the transcriptional profiles from two field-grown sugarcane genotype pools (each from 7 full-sib individuals), derived from a cross between two elite cultivars contrasting for Brix at two stages (early and late maturation). Up to now, we have 2,496 clones from each of the four SAGE libraries (high- and low Brix; early and late maturation), and we expect to generate 50,000 tags from each library. So far, for the high-sucrose genotype pool, 7,990 tags were isolated, comprising 5,377 unique sequences, from which 81% were detected just once. Annotation of tags was performed by matching tags to the SUCEST CAP3-clusters database via BLASTN. Despite comprehensive size of the SUCEST database, about 30% of the unique sequence tags lacked positive match (“no match“). Tag frequency distribution suggested a less redundant mRNA population for sugarcane internodes parenchyma as compared to a previously obtained SAGE leaf transcriptome. Among the 20 top most abundant tags, we found matches to putative genes corresponding to respiratory pathway (cytochrome precursors); structural proteins (ADF3); oligopeptides transporters; secondary metabolism (phenylalanine-ammonia-lyase, cinnamoyl-CoA reductase); stress-responsive proteins (superoxide dismutase, trypsin inhibitor, metallothioneins); and transcription, RNA processing and translation (zinc-finger transcription factors, glycine-rich RNA-binding protein, ribosomal proteins), with the highest similarity with Zea, Oryza, Hordeum and Arabidopsis genes. From the 20 most frequent tags, 4 had match to unknown plant proteins, 5 matched SUCEST clusters that have no hit with the GenBank, and the 9th most frequent tag has no SUCEST match. The many “no matches“ provide a new set of target genes potentially involved in sucrose accumulation process.
|3. IN SILICO Serial Analysis of Gene Expression Applied to the Sugarcane Transcriptome|
|Tercilio Calsa Junior
Based on the SAGE approach, a computational script was developed to generate virtual tags (~10 bp) from the SUCEST database, to assist in tag annotation and to investigate in silico gene expression patterns. A PERL script able to extract SAGE tags from SUCEST reads (over 50,000 reads) was developed, and it was used to analyze the SUCEST database by creating artificial concatamers for each library, followed by analysis at USAGE website (http://www.cmbi.kun.nl/usage/bin/login.cgi). A virtual sampling of gene expression profiles from the various tissues and/or conditions defined in each SUCEST library was possible for those reads containing a minimum poly-A tail (twenty -A or -T). Some potentially relevant biological comparisons were made between contrasting libraries (virtual tag sets), applying USAGE default parameters. Considering the virtual tags as representatives for gene expression profiles, their quantitative variation between tissues/conditions were tested, and some showed statistically significant differences based on the Audic & Claverie test, indicating possible involvements as transcription responses to environmental or cellular signals.
|4. Sugarcane Flowering Induction Genes Expression Analysis|
|Vagner Augusto Benedito, Danielle Scotton
Sugarcane flowering is an undesirable phenomenon for sucrose production. The identification of genes with major effect in responsiveness to flower induction would allow proposing ways to control flower induction in sugarcane cultivars. This project is using SAGE to compare transcription profiles from flower meristem at initial developmental stage of two sugarcane cultivar: an early inducible (‘SP87-432') and late or non-inducible (‘SP87-396'). Induction of flower meristem was followed by anatomical observations and by real-time RT-PCR using flower–specific genes. SAGE libraries were prepared at specific points of putative flower induction.
|5. Sugarcane Global Transcript Expression Study on Arbuscular Micorhyzal Development (Glomus clarum), Under Phosphorous Availability Influence|
|Raul Santin de Almeida, Silvio C. Christofoletti Jr.
About 70% of the world arable land presents low phosphorous (P) availability, restricting plant growth. Agriculture requires constant P fertilization using non-renewable sources. However, around 80% of the applied P becomes unavailable to plants due to interaction in the soil. High affinity P transporters are gene products responsible for P uptake under P deprivation conditions. Arbuscular mycorhyza (AM) symbiosis with higher plants favor the supply of nutrients by the fungus to the plant, especially P. Expression of high affinity P transporter genes in rice was shown to be modulated by P concentration and mycorhiza association. This project is investigating differential expression of sugarcane transcripts by SAGE in contrasting conditions for AM symbiosis (inoculated or not with Glomus clarum) and P dosage to identify possible cell signaling pathways which modulate and activate molecular responses of P homeostasis in sugarcane. Additionally, the spatial and temporal mode of phosphate transporter expression via promoter studies, and their possible biotechnological application.
|6. Evaluation of the Sugarcane Tolerance to Metals: Metallothionein and Phytochelatin Biosynthesis Pathway Enzymes Genes Expression|
|Maria Lorena Sereno, Deborah Sanae Nishimura, Raul Santin de Almeida, João Felipe Nebó Carlos de Oliveira
Heavy metals are elements with high density (>5 g.cm-3), naturally occurring in soils, but with increased concentration in the environment by anthropogenic activities. Heavy metals can be highly toxic (e.g. Mercury; Lead; Cadmium) or toxic at high concentration (Zinc and Copper). Phytoremediation is a technology based on the use of plants to remove or to transform pollutants in less toxic forms from the environment, at a lower cost than conventional physical-chemical methods. crop to be successfully adopted as a phytoremediator must be efficient in metal accumulation, yield large amounts of biomass and to be able to adapt to contaminated conditions, and sugarcane is recognized for high biomass production. We are currently investigating the control of expression of the chelating peptides metallothionein and phytochelatin. Results indicated that sugarcane plantlets are capable to maintain growth in concentrations up to 100 mM Cu or up to 500 mM Cd, while accumulating large amount of Cu and Cd, mainly in roots. Metallothionein genes expression was shown to be constitutive, with more expression of MT I and MT II, in leaves and roots. Metallothionein gene expression was poorly affected or not at all by Cu, but it was affected by Cd levels. Similar studies are underway to characterize the expression of genes encoding for the enzymes of the phytochelatin biosynthetic pathway: g-glutamyl-cysteine synthetase; glutathione synthetase; and phylochelatin synthase. Further, these same genes plus the metallothionein genes will also be studied by quantitative real-time RT-PCR. Our current results indicate the ability of sugarcane to tolerate and accumulate Cu and Cd, suggesting a potential application in phytoremediation of areas contaminated with heavy metals.
Nuclear Magnetic Resonance (NMR) is a powerful technique for the structural characterization of proteins and investigation of interactions between proteins and other biomolecules in solution. As such, NMR is an invaluable tool in the elucidation of biochemical mechanisms underlying plant development, among other biological processes, and is a methodology complementary to X-ray Crystallography for structural studies. For the initial part of the project we intend to identify a few targets "in silico", and subsequently clone and express the chosen target proteins for NMR studies.
We are particularly interested in proteins with nucleic acid binding activity, implicated in transcription and translational control in response to nutrient starvation, and also in proteins with unknown function expressed in conditions of nutrient starvation, for which a structural characterization may shed light on the cellular function.
We operate a multiuser NMR facility dedicated to Structural Biology, that at present has two Varian spectrometers, at 500 and 600 MHz, with a cryogenic probe for the 600MHz machine being installed at the end of August. The NMR facility is part of the Brazilian Synchrotron Light Laboratory, and is also open to users from all of South America.
The incresing availability of EST (Expressed Sequence Tags) sequences provides a potentially valuable source of new DNA markers. We aim to examine the SUCEST (Sugarcane expressed Sequence Tag Project) database and to develop EST-markers associated with economically important traits. EST-derived SSRs, EST-derived SCARs or EST-derived RFLPs are being used to enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in a sugarcane commercial cross. The EST markers are being screened among the 200F1 of the SP80-180xSP80-4966 mapping population. The integration of EST-derived markers will improve the map and, it renders possible to consider additional fine mapping of the genome. This will provide the means for developing ‘ideal markers’ associated with key QTLs and applications in comparative genomics.