II REUNION DE BIOLOGÍA VEGETAL 5-6 Noviembre...

II REUNION DE BIOLOGÍA VEGETAL 5-6 Noviembre 2007

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COMITE ORGANIZADOR

Patricio Arce P. Universidad Católica de Chile

Marlene Ayala

P. Universidad Católica de Chile

Virginia Garretón Austral Biotech & Universidad Santo Tomás

Rodrigo A. Gutiérrez (Coordinador)

P. Universidad Católica de Chile

Xavier Jordana P. Universidad Católica de Chile

Michael Handford

Universidad de Chile

Loreto Holuigue P. Universidad Católica de Chile

Erwin Krauskopf

Universidad Andrés Bello

Alejandra Moya Universidad de Talca

Ariel Orellana


Marlene Rosales Instituto de Investigaciones Agropecuarias

Herman Silva



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MEETING PROGRAM Monday November 5th MORNING 9:00 – 9:45 Registration and poster set up.

9:45 – 10:10 Welcome (Rodrigo Gutiérrez)

10:10-11:10 Opening Seminar Dr. Pam Green. Delaware Biotechnology Institute. University of Delaware. “Elucidating the small RNA Component of Plant Transcriptomes”. Host: Dr. Rodrigo Gutiérrez

11:10-11:30 Coffee break

11:30 – 13:00 Session 1: Genomics, Bioinformatics and Systems Biology

Chairs: Dr. Herman Silva and Dr. Marlene Rosales

11:30 - 12:00 Dr. Humberto Prieto, Instituto de Investigaciones Agropecuarias (INIA). Genetic transformation as a platform for gene function discovery in fruit crops.

12:00 Felipe Aceituno, P. Universidad Católica de Chile. Microarray data analysis reveals gene body methylation is a key factor for regulation of gene expression in Arabidopsis thaliana. Felipe F. Aceituno and Rodrigo A. Gutiérrez 12:15 Patricio Ramos, Universidad de Talca. Differential expression of genes involves in gravitropic response of Pinus radiata D. Don. Ramos , P., Moya, M., and Herrera.R 12:30 Loreto Prat, Universidad Andres Bello. Expression of aroma genes and accumulation of volatile compounds in cultivated strawberry (Fragaria x ananassa) and white strawberry (Fragaria


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chiloensis) during ripening. Prat, L., Marchant, L., Maldonado, J., Sanhueza, D., Agosin, E. and Silva, H. 12:45 Mónica Guzmán, Universidad Andres Bello. A first glance at the Eucalyptus globulus cold-activated regulatory pathway. Erwin Krauskopf, Susana Rasmussen-Poblete, Pablo D.T. Valenzuela, Maria Cecilia Gamboa.

13:00 – 14:30 Lunch break Monday November 5th AFTERNOON 14:30 – 15:30 Seminar

Dr. Hugo K. Dooner, Waksman Institute & Department of Plant Biology, Rutgers University. “Genome structure diversity and meiotic recombination in maize”. Host: Dr. Herman Silva

15:30 – 17:00 Session 2: Biochemistry and Molecular Biology Chairs: Xavier Jordana and Michael Handford

15:30 – 16:00 Dr. Jorge Casal. IFEVA - Universidad de Buenos Aires, Argentina. “Regulation of input-output relationships in phytochrome A signaling”

16:00 Hannetz Roschzttardtz, P. Universidad Católica de Chile. Regulation of SDH2-3 expression in Arabidopsis thaliana seeds. Binding studies of transcription factors to the promoter. Roschzttardtz, H., Vásquez, M., Gómez, I, Alonso, R., Vicente-Carbajosa, J., Araya, A., Jordana, X. 16:15 Francisca Blanco, P. Universidad Católica de Chile. Glutaredoxin C9 as a model gene for the study of the early response to salicylic acid in Arabidopsis thaliana. NPR1 dependence, promoter analysis and pathogen infection response. Blanco F., Herrera A, Cechinni N., Alvarez M, Laporte D, Holuigue L.


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16:30 Carlos Figueroa, Universidad de Talca. Differences in softening rates of strawberry fruits are related to differential pectin metabolism. Figueroa, C.R., Rosli, H.G., Civello, P.M., Pimentel, P., Gaete-Eastman, C., Herrera, R., Caligari, P.D.S., Moya-León, M.A. 16:45 Nilo Mejia, Instituto de Investigaciones Agropecuarias (INIA – La Platina). Positional candidate genes for seedlessness and berry weight in table grape. Nilo Mejia, Braulio Soto, Marcos Guerrero, Anne-Françoise Adam-Blondon and Patricio Hinrichsen.

17:00- 19:00 Poster presentations (Coffee and Beer) Tuesday November 6th MORNING

9:00-10:00 Seminar Dr. Nina Fedoroff. Willaman Professor of Life Sciences and Evan Pugh Professor, Biology Department and the Huck Institutes of the Life Sciences. The Pennsylvania State University. “The role of the Arabidopsis HYL1 protein in miRNA processing”.

Host: Virginia Garretón

10:00-11:30 Round Table

National policy for scientific and technological development. The Biofuels case study Session Chair: Virginia Garretón

10:00 – 10:30 Maria Olivia Recart, Subsecretaria Ministerio de Hacienda del Gobierno de Chile. 10:30 - 11:00 Dr. Nina Fedoroff, Science and Technology Advisor to U.S.

Secretary of the State Condoleezza Rice. Biology Department and Huck Institutes of the Life Sciences, The Pennsylvania State University.

11:00 - 11:30 Discusión.


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11:30:12:00 Coffee break 12:00-13:30 Session 3: Cell Biology

Chairs: Loreto Holuigue and Ariel Orellana. 12:00 – 12:30 Kristiina Himanen. Ghent University, Flanders Institute for Biotechnology, Belgium.. The arabidopsis ortholog of yeast BRE1 has a function in chromatin regulation during early organ growth. Himanen K., Fleury D., Cnops G., Nelissen H., Boccardi T., Inzé D., Van Lijsebettens M.

12:30 Dr. Gabriel León, Universidad Andrés Bello UDP-Glucose transporters AtUTr1 and AtUTr3 are partially redundant and essential for pollen development in Arabidopsis thaliana. Gabriel León, Francisca Reyes and Ariel Orellana 12:45 Ingrid Letelier, P. Universidad Católica de Chile. Expression of C/D-snoRNAs in Arabidopsis thaliana. Letelier I., Holuigue and Echeverría M. 13:00 Andrea Vega, P. Universidad Católica de Chile. Changes in gene expression in response to compatible viral infection in grapevine berries. Vega, A., Medina C., Arce-Johnson, P. 13:15 Elena Vidal, P. Universidad Católica de Chile. Nitrate:auxin interaction in the development of Arabidopsis thaliana roots. Vidal, EA., Araus, V. and Gutiérrez, RA.

13:30-15:00 Lunch break Tuesday November 6 AFTERNOON 15:00 - 16:30 Session 4: Plant Physiology

Chairs: Erwin Krauskopf and Alejandra Moya

15:00 – 15:30 Dr. Luis Corcuera, Universidad de Concepción. “A physiological approach to study the intriguing distribution of Notophagus dombeyi and Notophagus nitida”. Corcuera LJ, Reyes-Díaz M, Piper F, Alberdi M, Bravo LA.


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15:30 Andrés Zurita, Centro de Estudios Avanzados en Zonas Áridas - CEAZA. Assessment of salinity and boron toxicity on Arabidopsis thaliana root architecture: physiological responses and expression of target genes. C. Saguas, C. Cornejo, A. Milla, M-L Guillemin, K. Ruiz and A. Zurita. 15:45 Pablo Zamora, Universidad de Santiago de Chile Abiotic factors associated with the climate change and their effect on the Deschampsia antarctica biology. Pablo Zamora, Marcelo Ortega, Humberto Prieto, Gustavo E. Zúñiga. 16:00 Alvaro Castro, Instituto de Investigaciones Agropecuarias (INIA – La Platina). Evaluation for tolerance against Botrytis cinerea in a population of field lines of grapes cv. Thompson seedless genetically modified with antifungal genes. Álvaro Castro; Fernando Reyes1; Blanca Olmedo; Catalina Álvarez; Marisol Muñoz; Mario Pastén; María Antonieta Reyes; Manuel Acuña; Eduardo Tapia; Carlos Muñoz; Patricio Hinrichsen; Paola Dell´Orto1; Mike Moynihan1; Leonel Gonzalez2; Humberto Prieto. 16:15 Marcela Esterio, Universidad de Chile Molecular characterization of the genetic structure of Botrytis cinerea populations from table grapes (Vitis vinifera L.) in Chile. Marcela Esterio, Jaime Auger, Cecilia Ramos, M. José Araneda, G. Muñoz y M. Rosales.

16:30 - 17:10 Organizational Session – III Plant Biology Meeting Host: Organizing Committee.

[Open to all attendants] 17:10 - 18:30 Poster Session II 18:30 – 20:00 Meeting Reception


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INVITED SPEAKER

Elucidating the small RNA component of plant transcriptomes

Cheng Lu1, Dong-Hoon Jeong1, Fred Souret2, Monica Accerbi1, Sean Couglan3, Emanuele de Paoli1, Shawn Thatcher1, Karthik Kulkarni1, Manoj Pillay1, Kan Nobuta1, Ramesh Valliappan1, Shivakundan Tej 1, Blake C. Meyers1, and Pamela J. Green1 1Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA 2USB Corporation, 26111 Miles Road, Cleveland, Ohio 44128 3DuPont/Solae LLC PO Box 88940, St. Louis, MO 63188 Small RNAs such as miRNAs and siRNAs are a powerful regulatory force in most eukaryotes because they can function to shut off genes at multiple levels. Deep sequencing of small RNAs is an important step toward elucidating the impact of these molecules on individual genes and the genome as a whole. We have used Massively Parallel Signature Sequencing, 454 sequencing and Sequencing by Synthesis to sequence millions of small RNA sequences from wild type and several mutants of Arabidopsis including rdr2 [Lu et al., Science 309:1567,2005; Lu et al., Genome Res. 16:1276, 2006; http://mpss.udel.edu/at]. This led to the identification of a number of new miRNAs and targets including the mRNAs for two putative disease resistance proteins. To extend our knowledge of Arabidospsis small RNA regulation, we used DNA microarrays containing 2,500 small RNA oligos to examine expression in flowers, seedlings and roots. From rice, we sequenced four million small RNAs [http://mpss.udel.edu/rice] and identified more than 20 new miRNAs as well as several targets. Some of these miRNAs were conserved in other grasses like switchgrass and Brachypodium but none were found in Arabidopsis. Additional studies of Arabidopsis and rice small RNAs will be discussed, and a new comparative sequencing project to examine the small RNAs of more than 30 plant and algal species will be introduced. Funded by the NSF, DOE and the USDA.

http://mpss.udel.edu/at


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INVITED SPEAKER

Co-relation of transcriptional profiles and metabolic profiles to characterize the

development and ripening of grapevine berries Hugo Peña-Cortés1, Alvaro Cuadros1,3, Fernando Dorta1, Ingrid Ramírez1, Simón Ruíz2, Enrique González2, Fernando Poblete2, Camila Caldana3, Patricio Giavalescco3 and Lothar Willmitzer3. 1Centro de Biotecnología, Universidad Técnica Federico Santa María, Valparaíso, Chile 2Insituto de Biología y Biotecnología Vegetal, Universidad de Talca, Talca, Chile

3Max- Planck Institut für Molekulare-Pflanzenphysiologie, Golm, Germany. Different physiological and biochemical processes are involved in the fruit setting, development and ripening of grapevine berries. Diverse efforts using varied technologies are being applied with the objective to explain certain key biological stages of this fruit which could allow, in a near future, to improve the quality of grapevine berries and consequently the quality of wine. This study is part of a long-term work, which has as main goal the characterization of the biological processes involved in the development and maturation of this fruit. To gain information concerning the genes and metabolites involved in such processes, we are using genomics and metabolomics techniques in this study to establish a transcriptome and metabolomic profile co-relation data bank of Vitis vinifera. For this purpose, fruit samples of different development stages from distinct cultivars like Carmenère, Cabernet Sauvignon y Merlot are being analyzed at transcriptional level by using a qRT-PCR platform allowing the analysis of around 800 genes and at metabolomic level by using GC-MS, LC-MS y LTQ-Orbitrap. Samples of these cultivars have been collected every three days during two years for the whole growth period, starting by flowers and finishing with mature berries. From each sample, total RNA is being isolated for gene expression analysis and metabolites are extracted for metabolic profiling. By using this strategy we expect to establish a detailed analysis of the changes which occur at the level of gene expression and synthesis of metabolites during the development and ripening of grapevine berries. The progress obtained in this work will be discussed. FONDEF G07I1003


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INVITED SPEAKER

Genome structure diversity and meiotic recombination in maize

Hugo K. Dooner Waksman Institute & Department of Plant Biology, Rutgers University, Piscataway, NJ 08854, USA. The organization of the maize genome is remarkably polymorphic. The length and make-up of a given intergenic region can vary greatly among lines, mainly because of differences in the retrotransposon clusters that comprise most of the repetitive DNA in maize. However, other transposons, such as the classical McClintock transposable elements and the recently discovered Helitron elements, many of which carry multiple gene sequences, also contribute to this polymorphism. In our studies, we have focused on the well characterized bronze (bz) region, located in the short arm of chromosome 9 (9S). We have carried out a vertical comparison of 10 bz haplotypes from US Corn Belt and tropical inbreds, land races, and teosinte accessions. The extent of variation is remarkable. In pairwise comparisons, the percentage of shared sequences ranges from 25% to 84%. Chimeric haplotypes were identified that combine retrotransposon clusters found in different haplotypes. To investigate the effect of this great structural heterogeneity on recombination in maize, we have examined the distribution of recombination junctions in a 100-kb interval of 9S that contains two Helitrons and a large retrotransposon cluster, in addition to several genes. All the junctions are circumscribed to the gene space, where they are distributed in a highly nonuniform manner, creating recombination hotspots and coldspots. Chimeric haplotypes arise by recombination in the common gene space shared by heterozygous haplotypes. Recombination does not occur within retrotransposon clusters, explaining why genetic distances do not show more extreme variation in different genetic backgrounds. However, heterozygosity for large retrotransposon blocks does reduce recombination in adjacent genes. Therefore, local haplotype structure will significantly affect the correlation between genetic and physical distance for most intervals in maize.


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INVITED SPEAKER

Regulation of input-output relationships in phytochrome A signalling

Casal, J. IFEVA - Universidad de Buenos Aires

Light modulates plant growth and development. In particular, phytochrome A mediates two different photoresponses: the so-called very-low-fluence response (VLFR), which is required for germination when the seeds are briefly exposed to light during soil tillage, and the high-irradiance response (HIR), which is required for seedling de-etiotation and survival under dense plant canopies. The two response modes of phyA can be observed as discrete photobiological phases for some processes such as inhibition of hypocotyl growth and LHCB gene expression. The molecular mechanisms involved in the transition between the VLFR and the HIR with increasing frequency of phytochrome A excitation are not know. We will provide evidence in favour of a positive feed-back loop involving a transcription factor as a key component in the transition between VLFR and HIR.


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INVITED SPEAKER

The role of Arabidopsis HYL1 protein in miRNA processing

Zhicheng Dong, Liang Song, and Nina Fedoroff. Biology Department and Huck Institutes of the Life Sciences, 219 Wartik Laboratory, The Pennsylvania State University, University Park, PA 16803. We are studying the role of the HYL1 dsRNA-binding protein in miRNA biogenesis both in vivo and in vitro. We used hyl1 and dcl1 mutants to define the role of the proteins encoded by these genes in the biogenesis of AtMIR171a. The AtMIR171a gene has 3 transcription start sites. Two of the transcripts have 3 introns (the pre-miR171a is in the first intron) and the 3rd transcript initiates in the first intron. Pri-miR171a, but not the pre-miR171a, is much more abundant in hyl1 and dcl1 than in wildtype plants and it is the transcript that starts in the first intron that accumulates in both mutants, suggesting that the pre-miR171a is normally derived from the internally initiated transcript, which is rapidly degraded when the miRNA processing machinery is intact. To further define the role of the HYL1 protein in miRNA biogenesis, we developed an in vitro miRNA precursor processing assay using recombinant proteins. We expressed epitope-tagged DCL1, HYL1, and an additional protein reported to be involved in miRNA biogenesis, SE, in an insect cell expression system. We used the purified proteins singly and in combination to assess the requirements for processing synthetic pre-miR167b and pri-miR167b substrates in vitro. We find that DCL1 alone generates ca. 21 nucleotide digestion products using either substrate, but the cleavage is stimulated by the addition of HYL1 and SE. We analyzed the fidelity of cleavage by cloning and sequencing the oligonucleotide products. We find that HYL1 and SE each increase the fidelity of cleavage and appear to act synergistically.


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INVITED SPEAKER

The arabidopsis ortholog of yeast BRE1 has a function in chromatin regulation

during early organ growth Himanen K., Fleury D., Cnops G., Nelissen H., Boccardi T., Inzé D., Van Lijsebettens M. Ghent University, Flanders Institute for Biotechnology, Technologiepark 927, 9052 Ghent, Belgium. Histone modification and transcriptional activation are unconventional roles for ubiquitin E3 ligases that are usually associated with protein degradation. We have identified an Arabidopsis HCa-RING type ubiquitin E3 ligase as HISTONE MONOUBIQUITINATION protein1 (AtHUB1, and its homolog AtHUB2). AtHUB1 is an ortholog of human and yeast BRE1 protein. AtHUB1 monoubiquitinated histone H2B in vitro and genetic interaction studies confirmed that AtHUB1 and AtHUB2 act in the same pathway. Hub knockout mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth, suggesting that they have a role in organ growth. Kinematic analysis of leaf and root growth showed that hub1-1 mutation increased cell cycle duration in young leaves and resulted in reduced cell numbers and size. Flow cytometric analysis revealed a cell cycle block at G2-to-M transition and increased endoreduplication levels. Furthermore, transcript profiling of shoot apical meristems of hub1-1 showed that key regulators of the G2-to-M transition and cytokinesis as well as photosynthetic pathway were misexpressed in the mutant. Based on the mutant characterization, we propose a function for HUB1 in growth control through histone modifications that likely involves transcriptional regulation. We are currently performing structure-functional studies of AtHUB1 and AtHUB2 and TAP purifications are performed to identify the functional HUB protein complexes. Furthermore, the role of AtHUB1 in regulation of cell growth and division is assessed in overexpression and RNAi lines by physiological studies and transcriptional profiling.


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INVITED SPEAKER

A physiological approach to study the intriguing distribution of Nothofagus dombeyi

and Nothofagus nitida.

1Corcuera LJ, 2Reyes-Díaz M, 1Piper F, 2Alberdi M, 1Bravo LA 1Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción. Email: [email protected] 2Instituto de Agroindustria, Universidad de La Frontera, Temuco Nothofagus dombeyi (ND) and N. nitida (NN) are two closely related evergreens from the Chilean temperate rainforest that are rarely found growing together in spite of their ecological and phylogenetical proximity. ND presents a wide geographical distribution, including sites that often suffer frost, drought, and high radiation. Conversely, NN is restricted to more shady sites with oceanic climates, permanently humid soils, sometimes flooded or poorly drained. In this work we examine the hypothesis that seedlings of ND are more resistant than NN to environmental stress (frost, drought, radiation, and flooding). The LT50 were -12.8ºC and -6.2ºC for ND and NN seedlings, respectively. The incipient lethal soil water potentials were -3 and -2 Mpa for ND and NN, respectively. The photosynthetic apparatus in ND was typically of a sun plant, while that of NN was more similar to that of a shade plant. The energy dissipation mechanisms were more efficient in ND than NN under low temperature and high radiation at the seedling stage. The seedling stage of NN was more susceptible to photoinhibition induced by low temperature than ND. NN was more tolerant to waterlogging than ND. Based on these results we conclude that the contrasting resistance of ND and NN are consistent with their different geographical distribution. A model of how environmental factors affect their differential distribution will be presented. ND tends to grow in open areas with more radiation and extreme temperature and drought than NN. Conversely, NN tends to grow in more protected sites with less radiation, thermally more stable, and often subjected to waterlogging. FONDECYT 1030 663

mailto:[email protected]


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Oral Presentations Session 1: Genomics, Bioinformatics and

Systems Biology

Microarray data analysis reveals body methylation is a key factor for regulation of

gene expression in Arabidopsis thaliana.

Felipe F. Aceituno and Rodrigo A. Gutiérrez Departamento de Genética Molecular y Microbiología. Pontificia Universidad Católica de Chile. Microarray technology is a widely used approach for monitoring genome-wide gene expression. For Arabidopsis, there are over 1,800 microarray hybridizations representing many different experimental conditions in Affymetrix

TM ATH1 gene chips alone. This

tremendous amount of data offers a unique opportunity to infer new principles that govern regulation of gene expression in plants. In this study, we used bioinformatic methods to analyze publicly available data obtained with the ATH1 chip from Affymetrix. ATH1 hybridizations were normalized and filtered to eliminate low-quality hybridizations. We classified and compared control and treatment hybridizations and determined differential gene expression between control and treatment hybridizations. The largest differences in gene expression were observed when comparing samples obtained from different organs, on average ten-fold more genes were differentially expressed between organs as compared to any other experimental variable. This result indicates the Arabidopsis transcriptome is robust to most perturbations and it is established ontogenically. We defined “gene responsiveness” as the number of comparisons (control vs treatment) in which a gene changed its expression significantly. Genes with the highest and lowest responsiveness defined hypervariable and housekeeping genes respectively. Remarkably, methylation in the transcribed region was the feature that better distinguished housekeeping from hypervariable genes. Moreover, methylation in the transcribed region was inversely correlated (R2=0.8) to gene responsiveness on a genome-wide scale. This data supports a new functional role for gene body methylation as a key determinant for regulation of gene expression in Arabidopsis restraining the capacity of a gene to respond to stimuli. This work was funded by grants from: FONDECYT (1060457), FUNDACION ANDES (C14060/62) and NSF (DBI0445666) to R.A.G.. F.F.A. was funded by a Ph.D. CONICYT fellowship.


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Differential expression of genes involves in gravitropic response of Pinus radiata D. Don.

Ramos , P., Moya, M., and Herrera, R.

Laboratory of Vegetal Physiology and Molecular Genetic, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca. In the nature, conifer trees develop compression wood in response to gravitropic stimuli in the lower side of the stem. The genes involve and the molecular mechanisms under this phenomenon are still unknown. In this work we studied the expression of genes at early times from the stems bending-induced by inclination at 45º in radiata pine seedlings. The reorientation response is observed at 2,5 hours, we carry out a total RNA extraction at times 2,5 and 10 hours of induced the gravitropic stimuli from the stem of apical zone that response bending and, through the longitudinal cuts of the stem, we took samples from the upper and lower half. From those RNAs we generated substractive libraries (SSH) of ESTs, contained a total of 1200 clones with a size between 300 and 1500 pb. We estimated a redundancy value around of 40%. The analysis of the sequences by BLAST showed a differential expression of genes involves in intracellular transport, metabolism, cell wall formation and hormonal signals transduction among others. Support by Fondecyt 1071026 and DPI-UTALCA


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Expression of aroma genes and accumulation of volatile compounds in cultivated

strawberry (Fragaria x ananassa) and white strawberry (Fragaria chiloensis) during ripening.

Prat, L., Marchant, L., Maldonado, J., Sanhueza, D., Agosin, E.* and Silva, H.

Millennium Nucleus in Plant Cell Biology and Biotechnology and Plant Biotechnology Center Andres Bello University, Av. República 217, 837-0146, Santiago *Centro del aroma, Pontificia Universidad Católica de Chile Strawberry plants belong to the Rosaceae family and Fragaria genus. The cultivated varieties (Fragaria x ananassa) are hybrids from Fragaria chiloensis (native Chilean strawberry) and Fragaria virginiana (native of United States). Aroma volatiles contribute in a large extent to the overall sensory quality of the fruit. Research during the last decades has been dedicated to identification of volatile compounds present in cultivated strawberry but the study of aroma composition in Fragaria chiloensis is still limited, although his aroma is very characteristic and pleasant. The composition and concentration of fully mature fruit volatile compounds from cultivated strawberry (Fragaria x ananassa) and white strawberry (Fragaria chiloensis) are being investigated. Volatiles were isolated by two direct solvent extractions and analyzed by means of GC-FID and GC-O. We worked with 40,000 public nucleotide sequences of Fragaria x ananassa and 5,000 nucleotide sequences of Fragaria chiloensis. The sequences and the compounds information were used for the in silico reconstruction of the metabolic pathway. We are interested in characterize compounds unique to F. chiloensis or compounds that are present in a higher concentration when compared to F. x ananassa. We are also studying the expression of key genes that code for enzymes involved in the biosynthetic pathway of these compounds in three stages of development of both strawberries. Our preliminary results indicated that we were able to identify a set of genes that will participate in the biosynthesis pathway of some interesting aroma compounds. This research was supported by ICM P06-065-F and Proyecto regular UNAB DI-51-06/R


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A first glance at the Eucalyptus globulus cold-activated regulatory pathway

Erwin Krauskopf*1,2,3, Susana Rasmussen-Poblete1,2, Pablo D.T. Valenzuela 2,3,Maria Cecilia Gamboa 1,2 1. Universidad Andres Bello, Republica 252, Santiago, CHILE 2. Fundacion Ciencia para la Vida, Zañartu 1482, Ñuñoa, Santiago, CHILE 3. Millenium Institute for Fundamental and Applied Biology, Zañartu 1482, Ñuñoa, Santiago, CHILE [email protected] Eucalyptus globulus is the most important commercial temperate hardwood species in the world because its wood is particularly suited for paper making. In Chile, E. globulus commercial plantations are present from Concepción to Puerto Montt, the southern part of the country. Its growth rate and wood quality are negatively affected by low temperatures. As a first step towards overcoming these limitations, it is necessary to identify the repertoire of genes being expressed at low temperatures. A cDNA library was constructed from 3-month old E. globulus seedlings subjected to low temperature. In total, 9,913 randomly selected clones were sequenced, generating 8,737 curated expressed sequence tags (ESTs). The assembly produced 1,067 contigs and 3,879 singletons. Based on BLASTX analysis, 89.3% of the contigs and 88.5% of the singletons had significant similarity to known genes in the non-redundant database of GenBank. The unigene set was classified and analyzed according to gene ontology (GO) terms across functional categories. Overall, 541 unigenes were assigned to biological processes, 449 to cellular component and 493 to molecular function categories. From this cDNA library, we identified several genes that have been described in Arabidopsis as part of a regulatory pathway that is activated during cold acclimation. We have isolated these genes to establish if they function in a similar way as described for Arabidopsis thaliana. By real-time PCR we have established their relative expression throughout different time points of E. globulus seedlings exposed to 4oC and compared the results with the model proposed for plant Arabidopsis. The analysis of these experiments will be shown in this work.



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Oral Presentations Session 2: Biochemistry and Molecular

Biology

Regulation of SDH2-3 expression in Arabidopsis thaliana seeds. Binding studies of transcription factors to the promoter.

Roschzttardtz, H1., Vásquez, M1., Gómez, I1., Alonso, R2., Vicente-Carbajosa, J2., Araya, A3., Jordana, X1. Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile1, Laboratorio de Bioquímica y Biología Molecular, Dpto. de Biotecnología-UPM, E.T.S.I. Agrónomos, Madrid 28040, Spain2, Laboratoire de Réplication et Expression des Genes Eucaryotes et Rétroviraux, UMR 5097, CNRS et Université Victor Segalen-Bordeaux II, 33076 Bordeaux-Cedex, France 3. Our group has described that the SDH2-3 gene is specifically expressed in the embryo, during the seed maturation phase (Elorza et al., 2006, Plant Cell Physiol. 47:14-21). In this work, SDH2-3 promoter has been mutated and the modified promoters fused to the GUS reporter gene. The analysis of A. thaliana plants transformed with these constructions allowed us to conclude that mutations of ABRE (Abscisic acid response element) and RY cis regulatory elements affect SDH2-3 promoter activity. On the other hand, we have determined that SDH2-3 transcript levels decrease in abi3-5 and fus3-3 seeds, showing that ABI3 and FUSCA3 transcription factors are involved in SDH2-3 seed expression. Our results demostrate that the SDH2-3 gene is transcriptionally regulated by ABI3. Overexpression of ABI3 in transgenic 35S::ABI3 plants induces the ABA dependent expression of SDH2-3 in vegetative tissues. We will show the in vitro interaction of the transcription factors with the SDH2-3 promoter sequence. FONDECYT 1060485 and CONICYT AT-4040013.


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Glutaredoxin C9 as a model gene for the study of the early response to salicylic acid in Arabidopsis thaliana. Npr1 dependence, promoter analysis and pathogen infection

response

Blanco F1., Herrera A1, Cechinni N2., Alvarez M2, Laporte D1, Holuigue L1. 1 Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. 2 CIQUIBIC, Universidad de Córdoba, Córdoba, Argentina. Salicylic acid has a central role in activating defense gene expression in plants. According to the activation kinetics, those genes can be classified as early responding or late responding genes. Previous evidences suggest the existence of differential requirements for the activation of SA early response genes, such as the NPR1 co-activator and different cis-elements. Using microarrays and AFLP-TP, we identified 229 genes that are up-regulated after 2.5 h of SA treatment; 199 genes are NPR1-dependent and 30 are NPR1-independent. We used MIPS functional classification to group these genes in the most important categories of protein function. Later, for those two groups of genes we did an in silico analysis of the promoters sequences allowing the identification of putative cis-elements. We selected GRXC9 as a model gene, we analyzed the induction pattern, the NPR1- dependence, the dependence of de novo protein synthesis and the SA activation mechanism, using Northern blot analysis. We also studied the response of GRXC9 to bacterial infection. The intracellular SA levels were measured by HPLC in infected plants and in plants treated with exogenous SA. Finally, we did a promoter analysis of GRXC9 gene using transgenic plants carrying the PGRXC9-Egfp-GUS construction, we measured the reporter activity in SA treated plants and in infected plants with different pathogens. Financed by FONDECYT 1060494


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Differences in softening rates of strawberry fruits are related to differential pectin

metabolism. Figueroa, C.R.1, Rosli, H.G.2, Civello, P.M.2, Pimentel, P.1, Gaete-Eastman, C.1, Herrera, R.1, Caligari, P.D.S.1, Moya-León, M.A.1 1Laboratorio de Fisiología Vegetal, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile. 2Instituto Tecnológico de Chascomús, B7130IWA, Chascomús, Argentina. Fruit softening is certainly associated with a coordinated action of cell wall degrading enzymes. Several studies indicate that pectin solubilization increased during ripening of strawberry fruits, but the degree of pectin depolymerization depends on strawberry cultivar. In the present work, changes in solubilization and depolymerization of pectins during softening of Fragaria chiloensis and Fragaria × ananassa cv. Chandler fruits were analyzed. F. chiloensis fruits presented a higher softening rate than F. × ananassa between the large green and the turning fruit developmental stages. This phenomenon is associated in F. chiloensis with a great solubilization of covalently bound pectins. Instead, a high degree of pectin depolymerization is observed during softening of F. × ananassa. As Polygalacturonase (PG) and Pectate Lyase (PL) are probably involved in pectin metabolism, the expression of their genes and PG activity were measured. Two full-length cDNAs were isolated from F. chiloensis, FcPG1 and FcPL1, whose deduced polypeptide sequences showed a high conservation with PG and PL proteins of F. × ananassa, respectively. In F. chiloensis a higher level of PG activity and PG transcripts accumulation than in F. × ananassa were observed, while a great PL expression was observed in both species at the turning stage. In conclusion, the faster softening rate of F. chiloensis seems to be due to a fast solubilization of covalently bound pectins associated to a high and early PG activity. Acknowledgements to Fundación Andes C-13855/12, CIBS, CONICYT (Programa de Cooperación Internacional) and Anillo ACT-41 projects for financial support.


22

Positional candidate genes for seedlessness and berry weight in table grape

Nilo Mejia1, Braulio Soto1, Marcos Guerrero1, Anne-Françoise Adam-Blondon2 & Patricio Hinrichsen1 1 Unidad de Biotecnología, INIA La Platina, Av. Santa Rosa 11,610, Santiago, CHILE. 2

Unité Mixte de Recherches sur les Génomes des Végétaux, INRA, 2 rue Gaston Crémieux, 5708 91057 Evry-Cedex, FRANCE. Seedlessness, also called stenospermocarpy, and overall berry size are the principal quality traits in table grape, a major fruit commodity. In this work, a reverse genetic strategy was adopted to identify major genes involved in the control of seed and berry weight. Quantitative trait loci (QTLs) for seed and berry weight were mapped using a seedless x seedless derived progeny. Candidate Genes (CGs) were mapped in an Integrative Mapping approach (physical, genetic and in silico) using a Cabernet Sauvignon physical map and genomic sequences issued from the French–Italian Public Consortium for Grapevine Genome Characterization. Two CGs were mapped in the major seedlessness QTL identified in linkage group 18 (LG-18), a MADS-box transcription factor involved in ovule and seed development and a protein containing a FBOX domain, receptor for ubiquitination targets, key in hormone signalling. Two other CGs were mapped in berry weight QTLs, a putative PMEI (pectin methyl-esterase inhibitor) implicated in cell wall extension and fruit maturation in LG-2; and a putative SPINDLY, a negative regulator of gibberellin signalling in LG-8. The association of allelic variants with the corresponding phenotype is under evaluation in a core collection. These Positional Candidate Genes are suitable for the implementation of Gene Assisted Selection in breeding programs, and are also promising candidates to create improved lines by genetic transformation. Acknowledgements Partially funded by PBCT-Chile via “Consorcio BioFrutales” and a scholarship from Universidad de Talca and a Marie Curie Host fellowship for Early Stage Research Training, both to N.M.


23

Oral Presentations

Session 3: Cell Biology

UDP-Glucose transporters AtUTr1 and AtUTr3 are partially redundant and essential for pollen development in Arabidopsis thaliana

Gabriel León, Francisca Reyes and Ariel Orellana.

Millenium Nucleus in Plant Cell Biotechnology, Center of Plant Biotechnology, Andrés Bello University. The proper folding of glycoproteins in the endoplasmic reticulum (ER) requires continuous glucose trimming and re-glucosylation of unfolded glycoproteins. UDP-glucose is the substrate for this re-glucosylation cycle, and must be translocated from the cytosol into the ER lumen by UDP-glucose transporters. The Arabidopsis genome contains two homologous UDP-glucose transporters genes, AtUTr1 and AtUTr3. AtUTr1 is an ER-located protein and its expression is induced by ER stress albeit is not essential for plant development. Here, we show that AtUTr3 is an ER/Golgi localized UDP-glucose transporter ubiquitously expressed during plant development and induced by ER stress. Disruption of AtUTr3 has no obvious effect on growth and development. However, atutr1 atutr3 double homozygous mutant plants could not be obtained and the atutr1 atutr3 combination is not transmitted by pollen. Microscopic analysis shows that pollen development is altered, and several abnormalities were detected in mature pollen grains. Taken together, our results indicate that AtUTr3 mediates the uptake of UDP-glucose into ER and Golgi in an AtUTr1 complementary function, and demonstrate that the proper development of the male gametophyte requires at least one functional allele of these UDP-glucose transporters, establishing a gametophytic role for AtUTr1 and AtUTr3. Supported by FONDECYT 1070379 and PCB P06-065F


24

Expression of C/D-snoRNAs in Arabidopsis thaliana

Letelier I.1,2, Holuigue l,2 and Echeverría M.1

1LGDP, UMR-CNRS-5096, Université de Perpignan Via Domitia, France. 2DGMM, FCB, Pontificia Universidad Católica de Chile, Chile. The plant C/D-snoRNAs represents a large family of small non-coding RNAs that guide extensive modification of others RNAs, mainly rRNAs. Recent analyses of different plant genomes highlight their unique genomic organization and novel expression strategies. Nevertheless, mechanistic aspects of C/D-snoRNAs genes expression are still unknown. In this work, we characterize the C/D-snoRNAs gene expression in Arabidopsis. We identified new genes of C/D-snoRNAs in the Arabidopsis genome and for a selected group of these genes we characterized their precursor and mature transcripts and their promoter. Our results show an important transcript accumulation associated to cells or tissues in active proliferation. We determined that the accumulated transcripts are the mature C/D-snoRNA generated from a capped and polyadenylated precursor. For three of these precursors, the experimental determination of the transcription start site shows that two of these are polycistronic and one is monocistronic. The analysis of putative promoter sequences of the precursors reveals the presence of TATA, TELO and GCCCR motifs. To assess the functionality of these cis acting elements in the promoter sequences, we construct a C/D-snoRNA reporter gene and evaluate their expression in a wild type and 4 different mutated versions of the promoter sequence. We verified the functionality for the TATA and evaluated the requirement of TELO and GCCCR in vivo in Arabidopsis by sqRT-PCR. This work was supported by 1060494 FONDECYT, C00B03 and C03B03 ECOSCONICYT research grants.


25

Changes in gene expression in response to compatible viral infection in grapevine

berries.

Vega, A., Medina C., Arce-Johnson, P. Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas. Pontificia Universidad Católica de Chile. In viral compatible interactions, pathogens spread through all plant tissues causing disease without resistance response associated. Thus, these plants develop systemic diseases and chronic infections. However, susceptible hosts are not passive against viruses and they can set up a defense response. Systemic viral infection causes several changes in plant gene expression, underlying the symptom development and also controlling virus levels. Molecular events associated with viral compatible infections responsible for disease establishment and symptoms development are poorly understood. In this study, we analyzed viral infection in grapevines at a transcriptional level. With this aim, berries of Vitis vinifera red wine Cabernet Sauvignon cultivars naturally infected with GLRaV-3 were evaluated using real time RT-PCR. These analyses allow us to describe several genes that are induced or repressed in viral infected berries at different stage of development. Changes in gene expression associated with maturation, including processes of sugar metabolism and transport, anthocyanin synthesis, degradation of cell wall and hormone synthesis were mostly repressed. On the other hand, genes associated with cell defense are significantly induced during infection. This is the first study of gene expression in grapevine berries in response to viral infections and contributes to understand the changes triggered by viruses on grape berry physiology and its maturation. Acknowledgements: CONICYT, Innova 05CTE01-03.


26

Nitrate:auxin interaction in the development of Arabidopsis thaliana roots

Vidal, EA., Araus, V. and Gutiérrez, RA.

Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile Plant roots are highly plastic and adapt their growth and development in response to nutritional cues. Regulation of primary and/or lateral root growth represents a mechanism to optimize nutrient and water acquisition from soils. Nitrate, the main nitrogen source available in agricultural soils, stimulates primary and lateral root development. The molecular mechanism involved in controlling this phenotypic response is not completely understood, but it is known it involves modulation of plant hormone signaling pathways. Previous studies indicate that auxin signaling components are regulated by nitrogen treatments. Detailed time course experiments indicate that the expression of AFB3 is rapidly and strongly induced by nitrate treatments. AFB3 and other auxin receptors are known targets of miR393. To understand the role of miR393 and other small RNAs in the nitrogen response we used 454 sequencing of small RNAs from plants treated with nitrate/sucrose and control plants. Analysis of the sequence frequencies suggests that miR393 is induced by nitrate/sucrose treatments. This prompted the hyphothesis that auxin signaling modulation -by a signaling pathway involving miR393-, might be one of the mechanisms controlling root architecture in response to nitrate. Analysis of an AFB3 TDNA mutant (afb3-1) showed that AFB3 is key to lateral root development in response to nitrate supply, a response seen in wild type plants. Our results indicate that AFB3 auxin receptor has a specific role in regulating root architecture in response to nitrate availability in Arabidopsis. Further experiments to confirm the role of miR393 in the regulation of AFB3 by nitrate are underway. Acknowledgments : This work is funded by FONDECYT 1060457 and ICGEB CRPCHI0501 grants to Dr. Rodrigo A. Gutiérrez


27

Oral Presentations Session 4: Plant Physiology

Assessment of salinity and boron toxicity on Arabidopsis thaliana root architecture: physiological responses and expression of target genes

C. Saguas2, C. Cornejo2, A. Milla1, M-L Guillemin1, K. Ruiz1,3 and A. Zurita1* 1Centro de Estudios Avanzados en Zonas Áridas CEAZA- Universidad de La Serena Benavente 980, La Serena, Chile. 2 BSc. Agronomy, University of La Serena. Av. La Paz 1108, Ovalle, Chile. 3 Instituto de Investigaciones Agropecuarias. Centro Regional de Investigaciones Intihuasi, La Serena, Chile. * Corresponding author: [email protected] Current estimates indicate that 20-50% of all irrigated croplands are affected by high salt concentrations, resulting in major reduction of agricultural productivity. Atacama and Coquimbo regions in northern Chile are prominent examples of areas affected by both salinity and Boron toxicity. Abiotic stress affects plant growth and triggers diverse biochemical and physiological responses to acquire tolerance. Significant genomic resources have been established for the study of molecular responses on higher plants to salt toxicity and comprehensive microarray analysis of Arabidopsis transcriptome following NaCl exposure are available. To develop crop plants with enhanced tolerance to salinity by gene manipulation, a basic understanding of physiological, biochemical and gene regulatory networks is essential. Because roots are primary site of perception and damage for several types of waterlimiting stress and high salinity, roots are an attractive system for genomic and post genomics studies of abiotic stress responses. Moreover, the root sensitivity to stress modifies productivity of entire plant. To understand the effect of high salinity and boron toxicity in higher plants, different concentrations of NaCl and H3BO3 were applied to Arabidopsis thaliana grown on agar plates. The interactive effect of Boron toxicity and salinity stresses were investigated and several stress conditions were tested and compared to a control in 1-week-old seedlings. Increasing NaCl concentration, Boron concentration and the interactive effect of the two stresses were investigated on: (i) root length and architecture; (ii) chlorophyll concentration and physiological parameters; and (iii) the expression of target genes. To determine the effect of the different stresses on cell cycle, similar treatments were applied to Arabidopsis CYCB1;2 reporter line. Results of integrative studies will be discussed and the potential exploit for breeding abiotic stress tolerance in crop plants. ACKNOWLEDGEMENTS: INNOVA-CORFO project 05CR11PAT-19


28

Abiotic factors associated with the climate change and their effect on the

Deschampsia antarctica biology.

Pablo Zamora1, Marcelo Ortega1, Humberto Prieto2, Gustavo E. Zúñiga1 1 Biology Department, Chemistry and Biology Faculty, Universidad de Santiago de Chile. Santiago de Chile. 2 Instituto de Investigaciones Agropecuarias, La Platina Research Center, Santiago de Chile. Global climate has markedly changed in the past decades (EEA, 2004). It is expected that this event will cause not only temperature increasing but also changes in the rain regimes (IPCC, 2001). In that way, the whole process should generate more humidity at soil level, modifying its current biological activity. Some studies in the Artic area have experimentally demonstrated that the global change has really leaded to important impacts in their communities. However, the real impact in these changes is still less calculated and unpredicted when the Antarctic nest is analyzed.

Terrestrial communities of the Antarctic can survive to some of the most extreme conditions on the Earth, and in the same sense, they are more sensitive to the global change.

In this work, we have focused our interest in the native resource Deschampsia antarctica, and evaluated some of the abiotic factors involved in the global climate change, on the physiological and molecular-biological status of the species. Changes in the phenolic compounds and their relationship with photo- and oxy-protective abilities have been determined in samples obtained at in situ level. Modulation of their abundances and molecular composition of the most important compounds will be presented and discussed. All of them, are proposed as candidate protective compounds that should allow to the survival of this species in a highly altered environmental system (for instance with and extreme UV-B), occurring basically during its growing developmental stage.


29

Evaluation for tolerance against Botrytis cinerea in a population of field lines of

grapes cv. Thompson seedless genetically modified with antifungal genes.

Álvaro Castro; Fernando Reyes1; Blanca Olmedo; Catalina Álvarez; Marisol Muñoz; Mario Pastén; María Antonieta Reyes; Manuel Acuña; Eduardo Tapia; Carlos Muñoz; Patricio Hinrichsen; Paola Dell´Orto1; Mike Moynihan1; Leonel Gonzalez2; Humberto Prieto. Instituto de Investigaciones Agropecuarias, La Platina Research Center, Santiago de Chile. 1Fundación Chile, Santiago de Chile 2Agrícola Brown, Los Andes, Chile

Grey mold is among the major fungal diseases affecting Chilean grape crops. It is caused by the fungus Botrytis cinerea, causing large losses, both pre- and post-harvest. Chemical control of this disease is becoming less effective due to development of resistance by the pathogen. Furthermore, the application of fungicides has significant impacts on the environment and the human health. Novel strategies for control are based on biological pesticides, but due to their variable results are proposed as part of a system of integrated management.

The projects FONDEF D99I1001 and D01I1064, R&D were initiated to enable the genetic improvement of grapes using transformation. These projects resulted in a massive pipeline of genetic transformation for introduction of genes related to fungal resistance. Genes encoding enzymes that degrade cell walls of fungi (chitinases and glucanases) of the mycoparasitic fungus T. atroviride, were either licensed and isolated from native strains of Trichoderma. During the last five years, 5000 different transformation lines have been generated, with single- and tandem-gene constructs, leading to the establishment of 750 transgenic lines at field level. An ex-vivo test has been set up and applied for a 3-seasons period, allowing to the identification of 14 prototype lines, with an improved tolerance. Molecular characterization, agronomical behavior, biological responses, and up-coming commercial scale evaluation steps of this elite material will be shown and discussed as a very promising technology to be incorporated as a technological alternative to the grey mold control in Chile and overseas.


30

Molecular characterization of the genetic structure of Botrytis cinerea populations

from table grapes (Vitis vinifera l.) in Chile. Marcela Esterio1, Jaime Auger1, Cecilia Ramos1, M. José Araneda1, G. Muñoz2 y M. Rosales2.

1Depto. de Sanidad Vegetal, Fac. de Cs. Agronómicas, Universidad de Chile. Casilla 1004, Santiago – Chile; E-mail: [email protected]. 2INIA, CRI - La Platina y Carillanca. Chile is one of the main exporters and producers of table grapes from the Southern-hemisphere. “Gray mold” is the main fungal disease affecting this fruit crop, which is caused by the necrotrophic pathogen Botrytis cinerea. Molecular and biological studies undertaken during the last nine years have clarified the genetic structure of this pathogen in Chile. Our first results corroborated the presence of the three genotypes initially identified in France: vacuma, transposa and boty, which are differentiated by the presence or absence of transposable elements. The most prevalent genotype in table grapes correspond to transposa, with a parasitic character, it mainly infects berry grapes. Vacuma has a more saprophytic character and it is initially associated to grapevine leaves. The results also showed that transposa genotypes predominate in the sclerotial phase and that both, boty and vacuma, are highly sensitive to low temperatures. During the last two seasons we have analyzed: 1) the virulence of the fungal isolates recovered from different table grape cultivars and 2) the genotypic characteristics of the populations recovered from flower grapevine remainders. The results confirmed that 1) vacuma can also be associated to berry infections, and in this case seems to be important the presence of microlesions produced by excessive growth of the berry grape. We are now postulating that higher pathogenicity of vacuma in berry lesions would indicate that the transposable elements (Boty and Flipper), which are presents in transposa but not in vacuma genotypes, they could be intimately related with those genes involved in the first phase of the infection (our hypothesis under study); and 2) the presence of transposa, vacuma, boty and flipper in flowers remainders. This corresponds to the first report of the flipper genotype in Chile.



31

Poster presentations: Genomics, Bioinformatics and System Biology

The role of cytokinin signalling in the nitrate response of Arabidopsis thaliana

Naulin, P., Tamayo, K. and Gutiérrez, R.A. Departamento de Genética Molecular y Microbiología. P. Universidad Católica de Chile. Nitrogen is an essential macronutrient for plant growth and development. Nitrate is the main source of nitrogen available in agricultural soils and can act as a signal to modulate global gene expression in Arabidopsis thaliana. Despite the biotechnological relevance of understanding N-controlled gene expression, the molecular mechanisms involved in the plant response to nitrate are unknown. Research from our group and others suggest that plant hormones, and especially cytokinins, play a key role in the nitrogen response. As a first step to understand the role of cytokinin signal transduction in the nitrogen response, we evaluated the response to nitrate treatments of the hyposensitive cytokinin receptor mutants ahk2, ahk3, ahk4, as well as combinations of the double mutants of these receptors. Phenotypic analysis of the double mutants under different conditions of carbon and nitrogen indicate that cytokinin signal transduction is required for the primary root growth induced by nitrate. This growth phenotype is not a general root growth defect in the mutant, but it is manifested in the presence of nitrate. This result suggests that cytokinin act as a signal downstream of nitrate. Analyses of the expression of genes involved in the nitrogen assimilation pathway in one of the double mutants (ahk3/ahk4) by real time quantitative PCR suggests that cytokinin signaling is required for the normal regulation of this pathway in response to nitrate. Interestingly, none of the individual mutants showed phenotypic differences as compared to wild type plants. This result indicates that the nitrate effect is not mediated by a specific receptor, but is a consequence of a general defect in cytokinin signalling. Acknowledgements: This work is funded by FONDECYT (1060457) to R.A.G.


32

Coexpression gene networks to predict Arabidopsis gene function.

Alexander Vergara1-2-3, Ariel Orellana2, Rodrigo A. Gutiérrez3.

1.Doctorado BMCN, Facultad de Ciencias, Universidad de Chile. 2.Centro de Biotecnología Vegetal, Universidad Andrés Bello. Núcleo Milenio en Biología Celular Vegetal. 3.Departamento de Genética Molecular y Microbiología. Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile. Núcleo Milenio en Genómica Funcional de Plantas. DNA microarray technology is currently the most widely used approach for monitoring genome-wide gene expression changes in model organisms. Publicly available microarray data provides information on the expression of thousands of genes. This tremendous resource can be used to understand gene expression and to predict new properties of Arabidopsis genes. In this work we performed a global pair-wise linear correlation analysis of expression profiles using 1701 quality-screened AffymetrixTM ATH1 microarray and constructed gene coexpression networks. Analysis of the topology of these networks shows that they are fundamentally non-random, with scale-free degree distribution and small world characteristics. As a consequence, these networks show many genes with a low number of connections and a few highly connected genes called “hubs”. Hubs in scale-free networks are essential for the function of the network. However, many Arabidopsis hubs are genes without known function, suggesting we know little of these potentially key genes. In order to predict the potential function of these and other genes in the network, we developed a neighborhood counting based method that uses Gene Ontology (GO) terms to propagate the annotations. We obtained the best recall using a combination of the correlation values between the query and its neighbors. At the higher correlation cutoff, the more detailed predictions were obtained with a depth of 5 levels in the GO Directed Acyclic Graph (DAG) with recall values greater than 50%. Using this approach, for example, we can propose new genes involved in cell wall metabolism that are not predicted by other methods. Fondecyt-1060457-1070379-MNPCB-P02-009-F.


33

Poster presentations: Biochemistry and Molecular Biology

Study of differentially expressed tags during the development and ripening of the chilean strawberry fruit.

Pimentel, P., Salvatierra, A., Figueroa, C., Caligari, P.D.S., Herrera, R., Moya-León, M.A. Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile. The chilean strawberry fruit (Fragaria chiloensis L. (Duch.)) is a promisory fruit product for Chile. Its postharvest quality attractiveness is associated to the higher sweetness and aroma properties than the commercial strawberry. Ripening of strawberry fruit is associated with changes in texture, leading to an extensive softening which limits its post harvest life. During softening a partial dissembling of the fruit cell wall takes place which is associated with the coordinated action of many cell wall degrading enzymes. So far, we have investigated the role of key enzymes during softening identifying potential candidate genes for selection. In order to broad our knowledge on fruit softening we have generated several suppression substractive hybridization (SSH) libraries. By contrasting different developmental and ripening fruit stages, including large green (firm), large white and ripe fruit (soft), six SSH libraries were built containing 1809 differentially expressed cDNAs (ESTs). BLAST analysis of the ESTs revealed that 90% showed similarity to any known sequences within the plant kingdom. The global analysis of the cDNAs sequences revealed significant homology with genes involved in different plant processes such as primary and secondary metabolism, development, signal transduction, cell wall loosening and genes induced in response to plant growth regulators or environmental stress. The expression analysis of interesting cDNAs is under work. Acknowledgements to Fundación Andes C-13855/12 and Anillo ACT-41 projects for financial support and Conicyt for a doctoral fellowship to P. Pimentel.


34

Determination of the functional role of lycopene β cyclase (LCYB) in β-carotene biosynthesis in Daucus carota

Pizarro L, Stange C.

Laboratorio de Biología Molecular Vegetal. Departamento de Biología. Facultad de Ciencias. Universidad de Chile.

β-carotene, a carotenoid synthesized in plant plastids, is the precursor of abscisic acid and is involved in light harvesting in plants. In animals, β-carotene and α-carotene, are precursors for vitamin A and retinoic acid. The enzyme lycopene β cyclase (LCYB) is directly involved in the synthesis of β-carotene and α-carotene. In Solanum lycopersicum (tomato) two genes coding for LCYB have been identified. These are lcyb and cycb which are expressed in photosynthetic organs and fruits, respectively. Cycb has high sequence identity with the capsanthin capsorubin synthase gene (ccs) of Capsicum anuum (pepper) which codes for a CCS with proven LCYB activity.

Daucus carota (carrot) accumulates high levels of β-carotene in photosynthetic organs and in its modified root. There are several annotated lcyb cDNA sequences in D. carota. Recently, a complete cDNA sequence for ccs has been described in D. carota, which is predicted to code for a protein with LCYB activity.

To determine if lcyb has a redundant activity relative to ccs in D. carota, we studied the effect of post-transcriptional gene silencing of lcyb in this plant. To achieve this objective, we transformed carrot with the pBIN19-LCYB-AS binary vector in order to over-express a conserved antisense cDNA fragment of D. carota lcyb. We obtained three transgenic lines with different levels of silencing, as shown by semi-quantitative RT-PCR assays. Carotenoid concentrations measured by spectrophotometry and HPLC, showed that silenced lines have a higher production of total carotenoids than wild type plants. Levels of lutein, a derivative of α-carotene are particularly high, possibly as a result of a redirection of substrates in the pathway.

Acknowledgement DI 05/06 proyect.


35

Deteccion of virus in fragaria chiloensis crops

Salazar-Viedma, M.L., Lavandero, B., Rojas P., Sandoval, C., Caligari, P.D.S. Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile. Strawberries belong to the genus Fragaria, family Rosaceae. The name is derived from the Latin fragans, due to the fragrance of the fruits. The Chilean wild strawberry (Fragaria chiloensis L. Duch.) is a native species of Chile, which is one of the progenitors of the commercial strawberry (F. x ananassa), is widely distributed throughout the country in variable agroecological systems. White and red-fruited F. chiloensis forms exist, with the white fruited form being cultived in parts of South Chile. It is known for its intense fragrance and flavor. Strawberries are propagated vegetatively and are therefore susceptible to infection by viruses during plant propagation. More than 30 viruses diseases have been reported in Fragaria ssp. Improved virus detection and identification techniques are needed to allow efficient testing for quarantine, certification programs, identification of inoculum sources and production of healthy stock plants. The principal aphid-borne viruses infecting strawberry (Fragaria ssp.), e.g. Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) are reported to cause serious crop losses. In this study we have assessed Fragaria chiloensis (L.) Duch. collected at different sites in the south of Chile for virus. A RT-PCR method for the detection of all 4 viruses in combination with a plant mRNA specific internal control used as an indicator of the effectiveness of the RNA extraction was evaluated. Acknowledgements to DIAT, U Talca (Frutilla Chilena Integral), CIBS and Anillo ACT-41 projects for financial support.


36

Development and validation of a microarray-based detection system for grapevine viruses.

Escobar, P 2., Engel, E 2., Valdes, J 2., Fiore, N 1. y Valenzuela, P.D.T 2.

Facultad de Ciencias Agronómicas Universidad de Chile1 Fundación Ciencia para la Vida e Instituto Milenio de Biología Fundamental y Aplicada2. Zañartu 1482, Santiago, Chile [email protected]. Fruit exports are one of the main economic activities of Chile being grapevine among the most important species grown for table grapes and wine production. They are widely affected with viral pathogens that diminish the quality and quantity of the fruit obtained causing important economical losses. Early detection is difficult since disease symptoms may take several months to appear and severity depends on the susceptibility of the affected cultivar. Therefore, the adoption of quality control and government certification programs is of great importance to producers to avoid propagation of infected material. Towards this goal, we report the design of a comprehensive multi-viral detection system based on a microarray chip containing more than 500 DNA oligo probes to detect all viruses known to infect grapevines. These probes were designed using total or partial sequences available of all known grapevine viruses. This approach allows the detection of the already known viruses, and eventually new unknown viruses not explicitly represented on the array if their genomes have partial homology with the printed probes. Validation of the microarray using field samples with single and mixed infections of GLRaV-1, GLRaV-2, GLRaV-3, GFkV and RSPaV is shown. Results are compared with standard techniques such as ELISA and RT-PCR. Future versions of the chip will include probes to detect viruses from other fruit crops relevant to the export agro industry. Partially funded by CORFO-INNOVA 204-4040 and DI-UNAB 02-06/I.



37

Isolation and characterization of the Eucalyptus globulus ICE transcription factor

Susana Rasmussen-Poblete1,2, Monica Guzman2, María Cecilia Gamboa1,2, Pablo D.T. Valenzuela2,3 and Erwin Krauskopf 1,2,3

1Universidad Andrés Bello, Republica 217, Santiago. 2. Fundación Ciencia para la Vida, Zañartu 1482, Ñuñoa, Santiago. 3. Millenium Institute For Fundamental and Applied Biology (MIFAB), Santiago. Eucalyptus globulus is considered the most important temperate hardwood plantation species in the world. It is well known that E. globulus has the best combination of wood properties of commercial interest for the industry, especially for pulp and paper production. This tree has fast growth rates and the ability to adapt to a broad range of geographic locations, even though its growth rate diminishes due to frost conditions. In plants, signal transduction in response to cold conditions is mediated by a transcriptional cascade in which ICE (Inducer of CBF Expression) and probably other related proteins, induce the expression of CBF (C-repeat Binding Factors) genes, regulating the transcription of downstream genes that allow plants to withstand low temperatures. We isolated the full-length E. globulus ICE gene from a cDNA library built from E. globulus seedlings subjected to 4°C. The predicted EgICE protein has 560 aminoacids, exhibiting the same domains as three full-length ICE proteins described in GenBank. The expression of EgICE gene under cold (4°C) and drought conditions was determined using real time PCR. The results showed that this gene had a basal expression and that this expression was induced by both conditions during the first hour of treatment.


38

GONST4, a Golgi-localised nucleotide-sugar transporter of Arabidopsis thaliana,

transports GDP-fucose.

Miranda, J.P., Rámila, C., Febres, S., Ramírez, H.A., Caroca, R. and Handford, M. Laboratorio de Biología Molecular Vegetal, Departamento de Biología, Facultad de Ciencias, Universidad de Chile For the synthesis and/or modification of glycoproteins and polysaccharides in the lumen of the Golgi apparatus, specific transporter proteins are required for the import of nucleotide-sugars synthesised in the cytosol. We have identified two proteins from Arabidopsis thaliana GONST3 and 4, which possess the molecular characteristics of nucleotide-sugar transporters (NSTs) and which are related to NSTs transporting GDPsugars from other organisms. In addition, GONST3 and 4 are widely-expressed in the plant and are localised in the Golgi apparatus. Our work is focussed on determining the substrate specificity of these NSTs and on analysing their role in planta. In order to achieve the first goal, a 35S-GONST4-His vector was made (work in progress for GONST3). Using Agrobacterium, tobacco leaves were transiently transformed and expression of GONST4-His was confirmed by immunoblotting. After subcellular fractionation, microsomal vesicles isolated from GONST4-His expressing leaves were capable of transporting more GDP-[3H]fucose, but not UDP-[3H]glucose, than controlinfiltrated leaves. The specificity of GONST4 for other nucleotide-sugars is currently being tested. To address the second objective, we propose to lower and raise the expression levels of GONST3 and 4 and to analyse the phenotype(s) of the resulting lines. Constructs were made and Arabidopsis transformed to lower expression of GONST3 or 4 by RNAi. Moreover, in order to study the effect of over-expressing a GDP-sugar transporter in plants, Arabidopsis has been transformed with the GONST4-His construct and the presence of the insert has been confirmed at the molecular level. Current progress in our research will be presented. Funding: Fondecyt Iniciación 11060470.


39

Phylogenetic analysis of Fragaria chiloensis plants collected in Contulmo and its

response to Botrytis cinerea infection.

González, G.R1,3, Sandoval, C2., Moya, M1., Herrera, R1.

1. Laboratorio Fisiología vegetal, IBVB, Universidad de Talca., 2. Facultad Ciencias Agrarias, Universidad de Talca. 3. Centro Investigación Biotecnología Silvoagropecuaria. One of the most important post harvest problems in commercially cultivated strawberry is gray mold which it is caused by the fungus Botrytis cinerea Pers. This pathogen is widely distributed affecting more than 200 species most of them of economic importance. Fragaria chiloensis is characterized for having a higher pest and disease resistance spectrum, including gray mold. This, with other characteristics makes it an attractive specie for plant research. In this work we verified the identity of the Chilean strawberry plants collected from Contulmo using molecular tools, and verified their response to the infection with Botrytis cinerea. The F. chiloensis group was analyzed using Inter simple sequence repeat primers (ISSR). The results obtained with this molecular markers showed a high degree of similarity (66 and 100%) within the individuals. The molecular variance analysis (AMOVA) corroborated the identity of this group according to the Fst coefficient determined when the plants were compare to those the collection existing at the Universidad de Talca ( Fst: 0.0). This result indicate that the molecular markers utilized allows a fast an efficient identification. On the other hand, the individuals from the Contulmo group were classified according to their response to B. cinerea infection, 13 days after inoculation. The results obtained indicate statistical differences (p≤ 0.01) among the plants to infection. The severity of disease was divided in five categories: highly resistant (AR), resistant (R), tolerant (T), moderately tolerant (MT), and susceptible (S). Most plants were tolerant to infection (50%), but other 25% were moderately tolerant, indicanting that fragaria chiloensis has a genetic capacity to tolerate botritys infection. Acknowledgement: To Anillo ACT-41 project for finantial support.


40

Big vein disease (BVD) of lettuce: A biological and biochemical approach to

understand this disease Marlene Rosales, Carolina Araya, Lisset Román, Elizabeth Peña, Roxana Mora, Erika Salazar, Agustín Aljaro. INIA-CRI La Platina, Santiago. [email protected]. Big vein is an economically damaging disease complex of lettuce (Lactuca sativa L.) that occurs in all lettuce-producing areas. This disease involves two different viruses: Mirafiori lettuce big-vein virus (MLBVV) and Lettuce big-vein associated virus (LBVaV), both transmitted by the soil-borne fungus Olpidium brassicae. The disease is difficult to control since the resting spores of the fungus can persist for over 20 years in soil and because there are no major resistance genes available in commercial varieties of lettuce. A survey was conducted to measure occurrence and distribution of this disease in a total of 969 samples collected during years 2006 and 2007 in the major lettuce-growing areas of Chile (IV, V, RM, VI and VII regions). The results showed BVD is present in all regions surveyed with an overall incidence of 50%. Variety trials were conducted two consecutives years in Santiago and La Serena to evaluate the agronomic performance and genetic variation for big vein symptom expression in 127 lettuce varieties belonging to four different crop types (cos, butterhead, iceberg and leaf types). These were all infected by the viral agents associated to BVD, but there was a high symptom variation among lettuce groups and varieties. When we compared levels of symptom expressions and virus titer (determined through DAS-ELISA) in plants of similar genetic backgrounds, there was no statistical correlation. At present time we are studying the relation between virus genomic RNA and symptom development, and the possible role of some antioxidant enzymes (CAT, SOD, APX) in the course of the disease. This research was supported by projects: FIA-PI-C-2005-1-A-051 and FONDECYT INICIACIÓN Nº11060173.


41

Grapevines yellows in chile

Angela Gajardo1, Simona Prodan1, Samanta Paltrinieri2, Simona Botti2, Ana Maria Pino1, Jaime Montealegre3, Alan Zamorano1, Assunta Bertaccini2, Nicola Fiore1

1 Departamento de Producción Agrícola, Facultad de Ciencias Agronómicas, University

of Chile. 2 Dipartimento di Scienze e Tecnologie Agroambientali, Patologia vegetale, Alma Mater

Studiorum-University of Bologna, Bologna, Italy 3 Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas, University of

Chile Detection and identification of phytoplasmas associated with Chilean grapevines was performed. Symptomatic and/or asymptomatic grapevine plants were sampled and tested by nested PCR, that allows the detection of phytoplasmas in 30 out of 91 samples. Identification by RFLP analyses assigned the detected phytoplasmas to 16SrI-B, 16SrI-C, 16SrVII-A and 16SrXII-A ribosomal subgroups. Cloning and sequencing selected isolates allow the detection of genetic variability in 16SrXII-A Chilean strains. The finding of 16SrVII-A phytoplasmas is new for grapevine, while appear to be detected in different host species. Flavescence doree was not detected in Chilean grapevines. Infection with more than one type of phytoplasma is not uncommon in Chilean grapevines. Mixed infections between phytoplasmas and viruses frequently occur, that difficult the visual diagnosis. There is no evidence of epidemic spreading of yellows symptoms in the surveyed vineyards, nevertheless, future danger of 16SrXII-A epidemic spreading is not totally excluded. Survey to verify the presence and identity of insect vector or potential vector and also possible herbaceous weed hosts plants, acting as reservoir of phytoplasmas, are in progress. This work was financed by Fundación para la Innovación Agraria (FIA), project BIOT-BID-PI-C-2001-1-A-013.


42

Molecular characterization of chilean strains of Prunus necrotic ringspot virus

Nicola Fiore1, Simona Prodan1, Maria Carmen Herranz2, Frederic Aparicio2, Jaime Montealegre3, Alan Zamorano1,Vicente Pallás2 and Jesús Sánchez-Navarro2

1Departamento de Producción Agrícola, Facultad de Ciencias Agronómicas, University of Chile

2Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Avenida de los Naranjos s/n, 46022 Valencia, Spain

3Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas, University of Chile

Genes that encode the coat protein (CP) and the movement protein (MP) from 16

Chilean isolates of Prunus necrotic ringspot virus (PNRSV), obtained from different species of the Prunus genus: almond, plum, cherry, nectarin, peach, were amplified. Comparison of the sequences has been made. Analysing the evolutionary distance of nucleotide sequences and their deduced CP and MP amino acid sequences, all Chilean characterized isolates grouped into three PNRSV types, previously described as PV96, PV32 and PE5, being the first two groups prevalent. This suggests that difference does not exist between PNRSV Chilean isolates and those present in wide world fruit production areas. Research supported by CSIC proyect, framed-up by the collaboration between Chile and Spain.

Corresponding autor: N. Fiore Fax: +56 2 9785812 E-mail: [email protected]



43

Genetic transformation of two commercially important Prunus salicina varieties

using Agrobacterium tumefaciens infection of hypocotyl segments Carolina Urtubia3, Jessica Devia, Álvaro Castro, Mario Pastén, Pablo Zamora2, Carlos Aguirre3, Eduardo Tapia, Paola Barba1, Paola Dell`Orto1, Mike Moynihan1, Humberto Prieto. Instituto de Investigaciones Agropecuarias, La Platina Research Center, Santiago de Chile. 1Fundación Chile, Santiago de Chile. 2 Biology Department, Chemistry and Biology Faculty, Universidad de Santiago de Chile. Santiago de Chile. 3 Agronomical Sciences Faculty, Universidad de Chile, Santiago de Chile. In Chile, plums for fresh-fruit production are members of the species Prunus salicina. The main varieties are Angeleno and Larry Ann (24% and 23% of the total stone fruit production respectively). But there are threats to their successful production. In addition to the menace of the Plum Pox Virus (PPV), long distances to target markets have forced farmers and fruit producers to make considerable investments in PPV-free mother materials, and several post-harvest treatments. The latter are designed to avoid physiological disorders in stored fruits, i.e. progressive browning (internal browning) associated with woolliness in the mesocarp (flesh), and translucent breakdown just below the epidermal areas, together with flesh decomposition in areas surrounding the stones (gel breakdown). In this work we report the setup of a transformation pipeline in P. salicina, using fine-tuned adaptations of the A. tumefaciens and hypocotyl segments formerly described in Prunus domestica. Two varieties of both species, including Angeleno and Larry Ann, were successfully regenerated (with an optimal efficiency of approximately 12%) and stably transformed, generating 14 transgenic lines which were additionally characterized. Six transgenic lines were found to produce the transgenes mRNA, and the expression of green fluorescent protein, which was analyzed by confocal imaging of transgenic leaves in greenhouse kept trees.


44

N-terminal end of the Annona cherimola´s PPO allows to the sorting of the enzyme

into chloroplasts. Christian Montes, Humberto Prieto, Pablo Zamora1, Bruno Defilippi, Reinaldo Campos. Instituto de Investigaciones Agropecuarias, La Platina Research Centre, Santiago de Chile. 1 Biology Department, Chemistry and Biology Faculty, Universidad de Santiago de Chile. Santiago de Chile.

Cherimoya (Annona cherimola Mill.) fruit is an attractive candidate for food processing applications as fresh cut. However, it has a marked susceptibility to browning and this condition is mainly attributed to polyphenol oxidase activity (PPO). Recent works has allowed us the cloning, sequencing and experimental analysis of the full length Acppo gene. The predicted protein (acPPO) showed to have two candidate ATG starting codons, generating a confuse N-terminal analysis about the real compartmentalization of the enzyme by simple bionformatic processing of the sequences. A detailed functional analysis of the post-translational events involved in the final AcPPO sorting and cell distribution, was carried out by fusion of the two putative N-terminal signals found in AcPPO, to the green fluorescent protein (GFP) gene. Constructs either with the full-length (first ATG) or the second-half (second ATG) segment of the N-terminal peptide, were separately studied in transient expression assays. A. tumefaciens infections with clones harboring these constructs on N. tabacum leaves were then evaluated by confocal microscopy. The existence of a minimal sequence in this N-terminal segment of the gene, needed to direct AcPPO into chloroplasts was well established, supporting the idea that the cloned gene generates a protein that is sorted into these organelles.


45

Induction, maintenance, and genetic transformation of somatic embryogenic lines of

grapes cv. Thompson seedless using a semi-automatic airlift system

Eduardo Tapia, Álvaro Sequeida, Humberto Prieto. Instituto de Investigaciones Agropecuarias, La Platina Research Center, Santiago de Chile. High throughput systems for explants production are a key step of a genetic transformation pipeline. For grapes, production of thousands of somatic embryos is required for the different R&D activities (gene evaluation and silencing, and development of new varieties). The current somatic embryogenesis system used for explants supply has been the use of a solid-media based platform. A first scaling-up was conducted to replace it by a liquid-media based system. Slight hormone changes and the use of an adequate oxygenation speed, improved the specific growth speed (µ) from 0,023 d-1 (plates) to 0,049 d-1 (bottom flasks). Oxygen transference volumetric coefficient (Kla) was 213,4 h-1 (± 43,8) at 120 rpm, with an optimal temperature at 25°C and a 16 h photoperiod. Using this data, we designed a semi-automatic airlift bioreactor. Kinetic parameters and optimal conditions were then determined, with µ between 0,119 d-1 and 0,132 d-1, and volumetric productivities between 1,474 g/(dxL) to 1,595 g/(dxL) using pro-embrionary cells. When the reactor was inoculated with embrionary cells, µ was 0,040 d-1 and the volumetric productivity reached 0,482 g/(dxL). The optimal oxygen flow was 3 LPM at 25°C, generating Kla of 316,4 h-1 (± 90,3) and a gas retention coefficient of 0,348 in the downcomer section and 0,261 in the riser section; circulation and mixture times were 6,93 s and 80,43 s, respectively. The final explants were efficiently transformed by a conventional A. tumefaciens platform (solid-media based), as judged by gfp gene detection and expression in regenerated plantlets. This system has leaded to the reduction in the biomass duplication time from 30 days in the solid-media, into a 14 days system, with a considerable increasing of biomass yielding and ensuring explants flows for R&D.


46

Genetic diversity determination between Botrytis cinerea isolates growing on

Fragaria chiloensis and Fragaria x ananassa.

Valdes, J1, 2; Caligari, P1; Brevis, P2

1Instituto Biología Vegetal y Biotecnología, Universidad de Talca, 2Laboratorio de Microbiología, Fc Cs de la Salud, Universidad de Talca. Genetic diversity determination between Botrytis cinerea isolates growing on Fragaria chiloensis and Fragaria ananassa. Fungi attack is a mayor problem for strawberries (Fragaria spp). Among fungi species, Botrytis cinerea is the most frequent and harmful. This pathogen can grow at 4ºC on storing conditions, causing economic lose on harvested fruits. In the case of Fragaria x ananassa, there is some knowledge related to B. cinerea strains that attack frequently this strawberry. This strains can be differentiated by the presence of genetic transposable elements (known as transposons), therefore this elements are useful as molecular markers in F. x ananassa, but there is no knowledge about B. cinerea strains attacking the chilean strawberry, Fragaria chiloensis. Some authors suggest a correlation between two transposable elements, Boty and Flipper, and the pathogenicity or resistance of Botrytis to fungicides. In this work we identified the presence of these transposable elements some on isolates of B. cinerea afecting F. x ananassa and F. chiloensis. A total of 44 strains were isolated from F. chiloensis and 71 for F. x ananassa, from VII, VIII and XIX region of Chile. An additional strain isolated from grapevine was used as control (INIA, Santiago). Every strain analyzed contained at least one transponible element, demonstrating that B. cinerea isolates are very homogeneous. The similarity between fungi isolates was confirmed with ISSR (inter simple sequence repeat). Nevertheless, the statistical analysis of ISSR per geographic origin indicates that the originating strains of Quilicura are significantly different from rest of the localities, which can be considered like clones.


47

Characterization of two insertional mutants in the Arabidopsis thaliana SDH2-3 gene

Corvalán C., Fuentes I. and Jordana X.

Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Casilla 114-D, Santiago, Chile SDH2-3 is a nuclear gene encoding the iron-sulfur protein, a component of mitochondrial complex II. Other two nuclear genes, SDH2-1 and SDH2-2, encode the same protein but have a different expression pattern. While transcripts from SDH2-1 and SDH2-2 are expressed in all the organs of adult plants, the transcripts from SDH2-3 have been detected only during seed development. SDH2-3 transcripts appear during embryo maturation, are abundant in dry seeds and decline during germination. In contrast, transcripts of the other two genes can’t be detected during these stages. Those data, added to the fact that SDH2-3 possesses elements in his promoter that differ from the elements of SDH2-1/2, suggest a replacement of mitochondrial complex II subunits, SDH2-3 being replaced by SDH2-1 and/or SDH2-2 during germination. Due to its seed specific expression pattern, we evaluate the importance of this gene during embryo development and seed germination, using two insertional mutants in the SDH2-3 gene. Mutant seeds show a delay in germination in comparison with wild type seeds, but all of them germinate and seedlings develop normally. This is probably due to a compensation effect by the other genes and the replacement of SDH2-3 by SDH2-1 o SDH2-2. Recently, we obtain double sdh2-3/sdh2-1mutants, and preliminary results indicate a greater delay in germination of these mutants and a drastic diminution in the activity of de complex II in embryo’s tissue. Financed by Fondecyt Nº 1060485


48

Characterization of the promoter region of mitochondrial complex ii flavoprotein gene in Arabidopsis thaliana.

Carrasco, P1., Tapia, R1., Fuentes, D1., Jordana, X1. Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile1. In Arabidopsis thaliana two nuclear genes, SDH1-1 and SDH1-2, encode subunit I (flavoprotein) of mitochondrial respiratory complex II. A null mutant in SDH1-1 presents alterations in the development of both gametes, preventing the obtention of homozygous mutant plants. In contrast, homozygous mutant plants in SDH1-2 do not display an altered observable phenotype, suggesting that SDH1-2 is dispensable. The objective of this work is the characterization of the SDH1-1 promoter. For this purpose, two different lengths from the ATG upstream region, 434 pb (P1 construction) and 904 pb (P2 construction) were fused to the GUS reporter gene. Qualitative staining of the obtained transgenic plants showed that P1 is inactive, whereas P2 has weak activity in all tissues of adult plants. In addition, quantitative analysis of GUS activity demonstrated that there is an increase in promoter activity during post-germinative growth. Our results suggest that P2 construction, which includes all the intergenic region and part of the located upstream gene of SDH1-1, does not contain all the necessary elements for an efficient expression. On the other hand, to demonstrate that the phenotype observed in mutant SDH1-1 plants, is due only to the insertion in this gene, we decided to complement SDH1-1/sdh1-1 plants with a construct containing P2 fused to SDH1-1 cDNA. No complementation was observed, a result consistent with the weak promoter activity found for P2. Fondecyt regular project 1060485.


49

Genetic transformation of Solanum lycopersicum (Tomato) to evaluate genes

involved in abiotic stress tolerance Tania Timmermann Aranís, Consuelo Medina, Josefina Poupin and Patricio Arce-Johnson. Departamento de Genética Molecular y Microbiología, Laboratorio de Bioquímica. Pontificia Universidad Católica de Chile, email: [email protected] Tomato is one of the most important horticultural species for world agriculture and a convenient model for genetic analysis due to its short life cicle. We are interested in the study of genes related to abiotic stress tolerance (i.e: salt, cold and water stress). We have optimized in vitro culture techniques and a genetic transformation protocol for tomato (cv. Ailsa craig). We produced somatic organogenesis of this organism using cotyledons and specific concentrations of the phytohormones auxin and cytokinine. Cotyledons were used to obtain transgenic plants using Agrobacterium tumefaciens modified with a construction which has been used in other species as rice and tobacco to confer salt tolerance by overexpression of the enzymes that are involved in the Glyoxalase pathway (glyoxalase I and II). The transformed explants were periodically changed to fresh Murashige Skoog medium with BAP (Bencilaminopurine) and AIB (Indolbutiric acid) to induce shoot and root development, respectively. Antibiotic selective medium was used to identify possible transgenic plants, the transgeny of these was then verified by PCR of the foreign genes. We obtained twenty transgenic individual lines. The expression of the transgenes in six independent lines was evaluated by RT-PCR and quantified by real time RT-PCR. We are now evaluating the phenotype of these lines growing in high concentrations of NaCl. Acknowledgment: Consorcio Industria de la Fruta/ CORFO.


50

Analysis of mitochondrial complex ii function in Arabidopsis thaliana

Fuentes, D., Gomez, M.I. y Jordana, X.

Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile. Succinate dehydrogenase or complex II is an enzymatic complex that participates in the tricarboxylic acid cycle and the electron transport chain. In plants it contains two perypherical subunits (SDH1 y SDH2) and two integral membrane subunits (SDH3 y SDH4). SDH1, SDH2 and SDH3 are encoded by more than one gene. Our laboratory is interested in the characterization of mitochondrial complex II (SDH) function during plant development. For that purpose we use TTFA, a specific inhibitor of this enzyme. In this work, we show that SDH is important for postgerminative growth and saline tolerance. Furthermore, saline and osmotic stress induce an increase in transcript levels of two genes encoding for SDH subunits, suggesting that in stress conditions an increase of complex II activity may be necessary. Finally, heterozygous SDH1-1/sdh1-1 mutant plants show mitochondrial dysfunction and also a decrease in transcript levels of nuclear genes encoding mitochondrial complex I and II subunits. This suggests that a mitochondrial dysfunction may result in a coordinated decrease expression of nuclear genes encoding mitochondrial proteins. Supported by Fondecyt 1060485


51

Transcriptional regulation of the Phenylpropanoid Pathway in grapes: Isolation and

characterization of MyB, bHLH and WDR transcription factors

Matus, J.T.1, Poupin, M.J.2, Bordeu, E.1, Alcalde J.A.1 and P. Arce-Johnson2

1 Departamento de Fruticultura y Enología, Pontificia Universidad Católica de Chile. Santiago, Chile. Av. Vicuña Mackenna 4560. Santiago, Chile.

2 Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile. Alameda 340. PO Box 114-D. Santiago, Chile.

Within flavonoids, anthocyanins and polymerized proanthocyanidins are the two

most abundant pigments found in grape berry tissues. Their synthesis arises from the action of diverse enzymes which form part of the phenylpropanoid pathway. As described for model species such as Arabidopsis, maize and petunia, this pathway is controlled by the interaction of at least three families of transcription factors (TFs); Myb, Myc(bHLH) and WDR proteins. In grapes, only some Myb genes have been identified. In this study we have isolated and characterized different members of the three TFs families in order to establish their contribution towards the regulation of flavonoid synthesis. The repressor Myb4 was found to regulate negatively the latest enzyme of the pathway, while WDR1 and MycA1 contributed positively to the synthesis of anthocyanins once they were ectopically expressed in Arabidopsis or Nicotiana, respectively. Their sub-cellular localization was observed by means of GFP-protein fusions and their expression was screened in pigment producing organs, during inflorescence, berry skin and seed development. These findings allow screening TFs for biotechnological purposes towards manipulation of flavonoid pigments in grape, which contribute to wine quality and confer biomedical properties.

Acknowledgements: Consorcio de la Fruta y Consorcio del Vino (VINNOVA).


52

Research and ex situ collections of plant genetic resources preserved in Chile

Erika Salazar S.

Unidad de Recursos Genéticos, Instituto de Investigaciones Agropecuarias, Chile. [email protected] A study developed by INIA, during 2004-2005, showed that in Chile a total of 67,313 plant accessions are preserved under different ex situ conservation systems. 87.6% of these correspond to cultivated germplasm. The other 12.4% correspond to wild plants germplasm. 57% of the cultivated plants accessions conserved are cereals, legumes (16,3%), vegetables (8.6%) and forage crops (6,7%). Fruit trees, medicinal, aromatic and the ornamental plants are scarcely represented. 81% of the accessions are conserved in the INIA Germplasm Banks Network. Research works on the Chilean germplasm, in general, has been mainly related with prospecting, evaluation and conservation of materials of interest. The scientific study of the collections is a fundamental to define conservation strategies and to determine actions that can allow a sustainable use of the components of our genetic diversity. The development of new products and industries using the Chilean flora genetic resources must constitute an objective of its valuation. Of 5,801 species reported as native of Chile, approximately 10.3% have reported use. Validation of this local knowledge -through identification of its principles and/or active molecules, development of bioassays, agronomic and genetic characterization for the development of new varieties or the direct commercial use- is limited. On the other hand, the basic knowledge like morphological and biological description of species of great part of the Chilean biodiversity still been fragmentary and limited. Taxonomic, reproductive, genetic, biochemical and physical-chemistry studies of the collections require the interaction of different research groups with experience in several analytical and statistical techniques. For a suitable valuation of the Chilean germplasm, the interaction between experts in ex situ conservation with specialists in areas like the biotechnology, chemistry, taxonomy, biology, genetics and biostatistics is essential.



53

Chimeric Bacillus thuringiensis cry1 gene expression on tomato plants (Solanum

lycopersicum), to improve control of tomato moth (Tuta absoluta)

Alejandro Vega-Santander, Lorena Niedmann y Luís Meza-Basso Instituto de Biología Vegetal y Biotecnología. Universidad de Talca. Tomato moth (Tuta absoluta Meyrick, Lepidóptera:Gelechiidae) is a mayor pest for tomato (Solanum lycopersicum) cultures in Chile. T. absoluta is able to penetrate leaves and fruits, escaping toxic effect of substances unable to diffuse to vegetal tissues. This work intends to make transgenic tomato plants expressing Cry1 proteins, toxic for lepidopterans, on tissues susceptible to damage by tomato moth. As strategy, a chimeric cry1 gene, composed of the toxic fraction sequence of two different cry1 genes, will be cloned on chloroplast. From ten native Cry1 producing strains of Bacillus thuringiensis (Bt) evaluated to test toxicity against T. absoluta, two strains were selected, showing higher toxicity than commercial B. thuringiensis insecticide DIPEL. The toxic action was due by two different cry genes, which were subsequently isolated, sequenced and expressed on an acrystraliferous Bt strain. A second stage comprised the construction of a chimeric cry1 gene, coding for both toxic fractions of original Bt genes. An ongoing third stage will consist on the cloning of chimeric cry1 gene on tomato chloroplast genome, and the evaluation of toxic protein levels and in planta T. absoluta mortality.


54

Control of Tuta absoluta, a tomato moth, using the synergism between a chitinase and a native Cry1Ab endotoxin of Bacillus thuringiensis co-expressed in tomato

plants (Solanum lycopersicum)

Lorena Niedmann Lolas, Alejandro Vega-Santander y Luis Meza-Basso Instituto de Biología Vegetal y Biotecnología, Universidad de Talca Despite the success of commercial Bacillus thuringiensis (Bt) formulations, they have disadvantages under field conditions. Attempts for using Bt against Tuta absoluta larvae have not been successful due mainly to larvae nourishing habit. Young larvae penetrate tomato leaves, fruits or stems, creating mines and galleries. In this way, larvae escape the toxicity of pesticides including Bt formulations. It has been shown that Cry proteins and chitinase can help to reduce the impact of this pest when sprayed on tomatoes, existing an apparent synergical effect when both ingredients are used. However, the use of this mixture at the field level is limited by different factors. However, a more efficient way to deliver de Bt toxins and chitinase can be achieved by genetically engineering tomato plants producing both active components. To accomplish the main objective, native Bt isolates from our collection were evaluated measuring their biological assays against T. absoluta larvae. The LT50 values for each Bt strain were determined. Two strains, indexed as LM-033 and LM-012, performed the lowest LT50 (123 and 98 hours, respectively) at the lowest toxin dose (1.56 µg/ml). Assays showed that these isolates were more toxic than reference strain Bt var. kurstaki. The gene family type was determined by means of a PCR-based analysis using specific primers. Diverse cry genes (cry1Aa, cry1Ab, cry1Ac, cry1D, cry1F and cry3) were found. One cry1Ab gene was selected, cloned and sequenced for further plant transformation experiment. On the other hand, a chitinase gene was isolated from a Trichoderma strain and cloned in an expression vector. Biological activity of the recombinant chitinase was tested by monitoring growth inhibition of colonies of Botrytis cinerea. Current research is devoted to built a chimeric construct consisting of the Bt and a chitinase gene under the control of a RuBisco small subunit promoter.


55

The role of cytokinin signalling in the nitrate response of Arabidopsis thaliana

Naulin, P., Tamayo, K. and Gutiérrez, R.A.

Departamento de Genética Molecular y Microbiología. P. Universidad Católica de Chile. Nitrogen is an essential macronutrient for plant growth and development. Nitrate is the main source of nitrogen available in agricultural soils and can act as a signal to modulate global gene expression in Arabidopsis thaliana. Despite the biotechnological relevance of understanding N-controlled gene expression, the molecular mechanisms involved in the plant response to nitrate are unknown. Research from our group and others suggest that plant hormones, and especially cytokinins, play a key role in the nitrogen response. As a first step to understand the role of cytokinin signal transduction in the nitrogen response, we evaluated the response to nitrate treatments of the hyposensitive cytokinin receptor mutants ahk2, ahk3, ahk4, as well as combinations of the double mutants of these receptors. Phenotypic analysis of the double mutants under different conditions of carbon and nitrogen indicate that cytokinin signal transduction is required for the primary root growth induced by nitrate. This growth phenotype is not a general root growth defect in the mutant, but it is manifested in the presence of nitrate. This result suggests that cytokinin act as a signal downstream of nitrate. Analyses of the expression of genes involved in the nitrogen assimilation pathway in one of the double mutants (ahk3/ahk4) by real time quantitative PCR suggests that cytokinin signaling is required for the normal regulation of this pathway in response to nitrate. Interestingly, none of the individual mutants showed phenotypic differences as compared to wild type plants. This result indicates that the nitrate effect is not mediated by a specific receptor, but is a consequence of a general defect in cytokinin signalling. Acknowledgements: This work is funded by FONDECYT (1060457) to R.A.G.


56

Regulation of complex II (succinate dehydrogenase) during Arabidopsis

thaliana seed development.

Vasquez, M1., Roschzttardtz, H1., Gómez, I1., Fuentes, I., Jordana, X1. Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago, Chile.1. Complex II or Succinate dehydrogenase catalyzes oxidation of succinate to fumarate and reduction of ubiquinone to ubiquinol. Three genes, SDH2-1, SDH2-2, SDH2-3, encode for the iron-sulphur protein in Arabidopsis thaliana. Previously, we have determined that the SDH2-3 gene is expressed only during seed development and is regulated by transcription factor from the B3 family which are involved in regulation of gene expression during seed development. Until now, Arabidopsis ABI3 protein has never been found to directly bind DNA, and it has been postulated that ABI3 is recruited by interaction with bZIP transcription factors. In this work, we show that overexpression of bZIP10 and bZIP25 in transgenic Arabidopsis plants is able to induce the expression of typical seed genes, like At2S3 and Em6, in vegetative tissues. In contrast, SDH2-3 is not induced in these plants. Using transgenic plants carrying SDH2-1 and SDH2-2 promoter fusions to GUS, we show that these promoters have weak activity in seeds when compared to the SDH2-3 promoter or to the same promoter in vegetative tissues. In lec2 embryos, mutated in the B3 transcription factor LEC2, succinate dehydrogenase activity drastically decreases in cotyledons. Altogether, our results suggest that SDH2-3 is more important than SDH2-1 and SDH2-2 during seed development and/or germination


57

Characterization of an Arabidopsis lectin like protein (LLP) induced by salicylic

acid

Armijo, G., Salinas, P., Blanco, F., Tapia, J., and Holuigue, L. Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Salicylic acid (SA) is a crucial hormone for the establishment of the hypersensitive response (HR) and the systemic acquired resistance (SAR) to pathogens in plants. In previous work carried out in our laboratory, a group of genes activated by SA was identified in seedlings of Arabidopsis thaliana treated with this hormone. Within this group, a gene named llp (At5g03350) coding for a lectin like protein, was identified as highly responsive to SA. The product of this gene (LLP) has not been associated to any biological function; it has a carbohydrate binding domain and it is predicted to be located in the plant cell endomembrane system. We analyzed the induction pattern of llp by SA, the dependence of the coactivator NPR1, the dependence of de novo protein synthesis and the SA activation mechanism, using Northern blot analysis. Our results indicate that llp is early activated by SA by a transcriptional mechanism mediated by NPR1. We also analyzed the intracellular destination of LLP, by transiently expressing the LLPGFP fusion protein in tobacco leaves. As expected, LLP-GFP is located in the periphery of the plant cell, either in the plasma membrane or in the cell wall. The llp gene is strongly activated in Arabidopsis leaves infected with the avirulent bacteria Pseudomonas syringae pv tomato (avr Rpm1). We are currently evaluating the role of this protein in the defense response to pathogens, by using mutants and overexpressors lines for this gene. Financed by FONDECYT 1060494


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RNA interference as a strategy for resistance against Grapevine fanleaf virus

Bruno, C., Valenzuela, P.D. T.

Fundación Ciencia para la Vida e Instituto Milenio de Biología Fundamental y Aplicada, Av. Zañartu 1482, Santiago, Chile.

Grapevine fanleaf virus is one of the most important and widespread virus that affect Vitis genera, causing important economic losses due to the reduced quality of grape and productivity of vineyards.

The absence of natural resistance to this virus has carried to the development of pathogen-derived resistance strategies. RNAi appear as a novel mechanism of defense against pathogens in many organism, from the worm C. elegans to plants, as A. thaliana, and human cells. In latest model RNAi has been efficiently used to induce resistance to many viruses, in plants RNAi has been mainly used in functional genomics, nevertheless has been recently applied to produce plants resistant to many pathogens, including viruses.

Grapevine fanleaf virus is a (+) polarity virus, of the nepovirus groups. Its genome is encoded in two single stranded RNA. We chose three regions in the RNA2, varying in length from 300 to 500pb, corresponding to a gene responsible for its replication, the movement protein and the coat protein, respectively. These regions were amplified by PCR from a cDNA library of a Chilean isolate of GFLV (GFLV-Ch80), that has been completely sequenced, and cloned by homologous recombination in the binary vector pHellsgate2, which has been specifically designed for RNAi. Agrobacterium tumefaciens was transformed with these vectors and used to transform Nicotiana benthamiana explants by coculture. Transgenic plants were regenerated and selected in kanamycin medium, the insertion of the constructs in transgenic plants genome was confirmed by PCR, obtaining a total of 50 transgenic plants. The expression of transgene as long RNA and the accumulation as siRNA were analyzed, as well as their resistance against infection with GFLV.


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Poster presentations: Cell Biology

Role of auxin transport in growth inhibition during the defensive response in Arabidopsis thaliana.

Santiago, M. and Silva, H.

Millennium Nucleus in Plant Cell Biology and Biotechnology and Plant Biotechnology Center, Andres Bello University, Av. República 217, 837-0146, Santiago. The defence against pathogens in plants is associated to the recognition of the pathogen by the plant. There are two forms of recognition: general and specific. In the first one, the plant recognizes common motifs or PAMPs (Pathogen Associated Molecular Patterns) and in the second, the plant recognizes the avirulence factors, specific to each pathogen. One of the main bacterial PAMPs is flagellin, a structural component of the flagellum. This protein has a conserved domain of 22 amino acids, denominated flg22, which is able to generate the same response that the complete protein. The defensive response developed by exposing Arabidopsis thaliana to flg22 is characterized by an increase in reactive oxygen species (ROS), accumulation of callose, increase in the expression of PR (pathogenesis related) genes and a strong inhibition of plant growth. It is believe that the growth inhibition is due to the low use of energy to favour the defence and/or a diminution of the colonisable tissue by the pathogen. Experimental evidence has demonstrated that the treatment with flg22 diminishes the transcript of at least three auxin`s receptors and other genes associated to the auxin signalling, like PIN3 efflux transporter. These data suggest that auxin transport and accumulation could be altered in Arabidopsis thaliana during the defensive response against flg22. Our main goal is to study the role of auxin transport during the Arabidopsis defensive response. Preliminary results indicate that development of secondary roots is inhibited in seedlings treated with flg22, phenotype that is also showed in seedlings treated with the auxin transport inhibitor NPA. This research was supported by ICM P06-065-F and IFS C/3387-1


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Molecular and biological behavior of the transgenic resistant C5 plum upon challenges with five Chilean isolates of the Plum Pox Potyvirus (PPV)

Wendy Wong3, Álvaro Castro, Catalina Álvarez, Mario Pastén, Pablo Zamora2, Marlene Rosales, Paola Dell´Orto1, Mike Moynihan1, Humberto Prieto. Instituto de Investigaciones Agropecuarias, La Platina Research Center, Santiago de Chile. 1Fundación Chile, Santiago de Chile. 2 Biology Department, Chemistry and Biology Faculty, Universidad de Santiago de Chile. Santiago de Chile. 3 Agronomical Sciences Faculty, Universidad de Chile, Santiago de Chile. The genetic transformation approach to get new varieties has a direct example by application of gene silencing introducing viral gene sequences (often coat proteins (CP)) into the host genomes. However, recalcitrant species as in the genus Prunus, this technology seems to be more restricted. The only transformation example fully confident until now has been the generation of the C5 event (P. domestica), which has been modified by insertion of the full length CP gene of the Plum Pox Potyvirus (PPV). The importance of PPV has encouraged several C5 field trials (North America and Europe), showing promising results and resistance to several PPV isolates of D and M strains. Recently, has been proposed that the induced protection in C5 is due to the silencing process. Overseas validation of the C5 event is a fundamental to create confident data set of results. In that line, C5 plums were multiplied and evaluated for challenging with five different PPV Chilean isolates (strain D). Sticks of C5 generated in vitro were micro-grafted into a population of previously chip-budded, PPV positive, Prunus insititia L. hosts. In planta viral loads (in rootstocks and scions) were evaluated during two different seasons. Symptomologies, ELISAs, Western blots, immunocapture-RT-PCRs and “region-specific” RT-PCRs were carried out. Reinforcement of the previous experience with C5, with a complete resistance phenotype to the Chilean isolates mediated by gene silencing has been found.


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AtUTr1 and AtUTr3 expression under different stresses suggest a common

transcriptional regulation with genes involved in protein quality control

Adrián Moreno and Ariel Orellana. Millenium Nucleus in Plant Cell Biotechnology, Center for Plant Biotechnology, Andres Bello University. In the protein quality control process, UDP-glucose is required to the reglucosilation of misfolded proteins by the UGGT enzyme. This modification allows the entry of misfolded proteins to the Calreticulin/Calnexin cycle to help them to reach their correct folded structure. If this process failed, the misfolded proteins become accumulated in the ER and trigger the unfolded protein response (UPR). The most important consequence of this response is the up-regulation of ER chaperones. AtUTr1 and AtUTr3 are UDP-glucose transporters and ER residents. If these transporters are involved in the quality control process we can expect that their expression patterns should be similar to the expression patterns of the ER chaperones genes involved in this process. As an experimental approach we analyzed the expression of several ER chaperones, AtUTr1 and AtUTr3 by means of real-time RT-PCR under different stress conditions. Our results show that the expression pattern of AtUTr1 and AtUTr3 is similar to the ER chaperones under different stress conditions. In silico analysis reveal the presence of common cis-acting elements in the promoter region of AtUTr1, AtUTr3 and ER chaperones. In addition, we are analyzing the expression level of ER chaperones in mutant plants that harbor only one functional copy of either AtUTr1 or AtUTr3. We hypothesize that in these mutants the low levels of UDP-glucose in the ER will enhanced the accumulation of misfolded proteins due the lack of reglucosylation and this accumulation will trigger the UPR. Currently, we are performing the analysis of the expression level of the ER chaperones in the mutant plants. Supported by Fondecyt, 1070379 and Millennium Nucleus PCB P06-065F


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Heterologous expression analysis of Arabidopsis secreted proteins that exhibit polar

destination

Rodriguez-Furlán, C., Orellana, A. Millenium Nucleus in Plant Cell Biotechnology, Center of Plant Biotechnology, Andres Bello University, Santiago, Chile. The plant cell wall is a major determinant of cell morphogenesis. Cell enlargement depends on the tightly regulated expansion of the wall, which surrounds each cell. Several key proteins involved in cell wall synthesis, remodeling and assembly must be secreted specifically to the enlargement zone in a polarized fashion. However, the identities of these proteins are still poorly characterized mainly because these studies within the plant cells possess a number of disadvantages. Since the molecular mechanisms for correct targeting of proteins in eukaryotic cells are highly conserved, we propose a heterologous expression system to identifying plant proteins secreted asymmetrically. We choose a mammalian epithelial cell system that exhibit polar morphology, the MDCK cells. Here we analyze the polarized secretion of AtPRP3 and AtPRP1, which are expressed in root hairs at the apical tip and at the base respectively. The MDCK cells expressing either AtPRP3 or AtPRP1 were grown in permeable supports that allowed us to analyze separately the apical from the basal cellular medium. The results showed that AtPRP3 was sorted to the apical domain and that AtPRP1 was secreted in a non polarized way. We also treated the cells with 10 ug/ml tunicamycin to analyze the possibility that the apical sorting of AtPRP3 could depend of the N-glycan moites of these proteins. Our results suggest that the exocytic machinery involved in the targeting of AtPRP3 is an N-glycan-dependent pathway. We are performing additional experiments to homologate the N-glycan-dependency in the plant model. The use of an heterologous expression system to characterize the asymmetrical extracellular sorting of plant proteins will be discussed. Supported by PCB-MN P06-065F


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The Actin Cytoskeleton Plays a Role in Nuclear and Mitochondrial Movement in

Response to Blue Light

Fernanda Rodríguez, Andrea Morales and Lee Meisel. Millennium Nucleus in Biotechnology and Plant Cell Biology. Plant Biotechnology Center, Andrés Bello University. Photosynthetic organisms such as plants have developed diverse mechanisms in order to capture and utilize solar energy to realize complex metabolic processes. However, high intensity of certain wavelengths of light can cause irreversible damage to intracellular structures such as organelles. In order to avoid the damaging effects of high intense blue light (426-446 nm) chloroplasts have been shown to move away from this light source (avoidance response). We have shown previously that not only chloroplasts avoid blue light, but also mitochondria and nuclei. In order to decipher the mechanisms associated with nuclear and mitochondrial movement in response to blue light, we have analyzed the role that the cytoskeleton plays in this avoidance response. Using diverse drugs that alter actin microfilaments and microtubules, we demonstrate that nuclear and mitochondrial movement in response to blue light is an actin dependent process. (ICM P02-009-F, ICM P06-065-F, UNAB Proyectos de Investigación DI-44-05/R)


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Gamma Adaptin-like proteins play a putative role in trafficking the syntaxin,

KNOLLE, in post mitotic cells

Daniela C. Urbina, Mauricio Valdivia, Ricardo Tejos and Lee A. Meisel. Millennium Nucleus in Biotechnology and Plant Cell Biology. Plant Biotechnology Center, Andrés Bello University. Plant cell polarity is intimately related to the polarized develop and growth of plant organs (apical: shoots, leaves and flowers; basal: roots). Polarized growth begins early in the embryonic stages of development, establishing two polarized axes which produce roots and aerial organs. Analyses of Arabidopsis embryonic mutants have related components of the secretory pathway with establishing plant cell polarity. It has been previously reported that the syntaxin, KNOLLE, is expressed and accumulated exclusively in cells undergoing division, and participates in polarized vesicle trafficking between the TGN (Trans Golgi Network) and the cell plate. Additionally, it has been demonstrated that when cell division is completed, KNOLLE is transported to the vacuole, where it is degraded. Using reverse genetic approaches and immunolocalization analyses, we present data that suggests that Gamma-like adaptins play a role in trafficking the syntaxin KNOLLE in post mitotic cells. (ICM P02-009-F. ICM P06-065-F, CONICYT PhD Scholarship and the Department of Developmental Genetics, Tübingen University, Germany)


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Poster presentations: Physiology

Browning in Annona cherimola fruit: role of polyphenol oxidase (PPO) and characterization of a coding sequence of the enzyme

Humberto Prietoa, Daniella Utzb, Álvaro Castroa, Carlos Aguirrea, Mauricio González- Agüeroa, Héctor Valdésc, Nicolas Cifuentesb, Bruno G. Defilippia, Pablo Zamorab, Gustavo Zúñigab and Reinaldo Campos-Vargasa* a Instituto de Investigaciones Agropecuarias, INIA, Chile. b

Facultad de Química y Biología, U. de Santiago de Chile. c

Facultad de Ciencias, Escuela de Agronomía, U. La Serena, Chile. * Corresponding autor: [email protected] Cherimoya (Annona cherimola Mill.) fruit is a candidate for fresh cut applications. However, cherimoya shows a marked susceptibility to browning. This condition is mainly attributed to polyphenol oxidase activity (PPO). The work carried out included the cloning of a full-length cDNA, an analysis of its properties, and linkage of its mRNA levels with enzyme activity in mature and ripe fruits after wounding. The results showed one gene different at the nucleotide level when compared with previously reported genes, but a well-conserved protein. Cherimoya PPO gene (Ac-ppo, GenBank DQ990911) showed to be present apparently in one copy of the genome, and its transcripts could be significantly detected in leaves, and less abundant in flowers and fruits. In addition, efforts to localize PPO in cherimoya’s cell indicated that the chloroplast would be the position for PPO protein. Analysis of wounded matured and ripen fruits revealed an inductive behavior for mRNA levels in the flesh of mature cherimoya after 16 h. Although the highest enzymatic activity was observed on rind, a consistent PPO activity was detected on flesh samples. A lack of correlation between PPO mRNA level and PPO activity was observed, especially in flesh tissue. This could be due to the presence of inhibitor(s) compound(s) in the flesh. To our knowledge this is the first report of PPO in cherimoya. (Funded in part by FONDECYT 1040011).



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Advances in Micropropagation of Belloto del Norte (Beilschmiedia miersii (Gay) Kosterm)

Contreras GE, Azúa-Bustos A, Prehn D

Facultad de Agronomía e Ingeniería Forestal, Departamento de Fruticultura y Enología, Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul, Santiago, Chile. Belloto del Norte (Beilschmiedia miersii) is an endemic species of central Chile mainly present between regions V and RM. In 1995 it was declared national monument and is presently considered in vulnerable category. The few remaining forests are fragmented due to farming and mining activities and show low natural regeneration rates. Here we show the first results of a protocol for in vitro propagation of Beilschmiedia with the purpose of contributing to the conservation and recovery of the species. Nodal explants of 1-3 year old green-house grown plants and of new growth from field adult trees were used for tissue culture initiation. Disinfection with anti fungi compounds Captan/Benlate (10 g/L each one) for 15 min., followed by a 30% v/v commercial chlorine bath for 15 min, and three rinses in sterile distilled water proved to be successful for young explants. Initial cultures of greenhouse explants showed contamination rates of 30%, whereas over 95% of adult field explants were lost due to contamination. Greenhouse grown plants were submitted to pest management to control fungi three weeks prior to establishment. Explants were established in full strength MS media with combinations of GA3, BA and IBA or basal DKW salts, respectively. MS medium with GA3 allowed growing young buds with normal leaf shape and growth. The addition of BA stimulated the generation of buds. The medium with basal DKW salts improved total number of buds, length of buds and number of leaves/bud in comparison with media with MS salts. DKW also reduced lenticels hypertrophy, a physiological disorder that affected around 30 % of the buds. Direct rooting of node segments in vitro in media supplemented with 1 and 4 mg/L IBA did not stimulate the generation of roots. The results serve as base for the development of a protocol for in vitro multiplication of Beilschmiedia miersii . Proyecto Recuperación Belloto del Norte, dirigido por H Gilabert y financiado por Angloamerican Chile.


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Molecular and physiological bases of aroma biosynthesis in apricot fruit (Prunus armeniaca L.)

Defilippi, B.G1., González-Agüero, M.1, Troncoso, S.2, Gudenschwager, O.1, Campos-Vargas, R.1.

1Instituto de Investigaciones Agropecuarias, CRI La Platina, Casilla 439-3, Santiago. 2 Facultad de Química y Biología, U. de Santiago de Chile, Av. Bernardo O’Higgins 3363, Santiago. Email: [email protected]

A salient genetic attribute of tree fruits is the unique blend of sugar, acid, phenolic and volatile components that determine their flavor. This complex genetic trait is manifested in ripe fruit through a complex interaction of metabolic pathways and regulatory circuits that results in the unique fruit flavor composition, a key to fruit consumption. Loss of flavor, particularly the aroma attribute, is a limiting factor in apricot quality. In spite of its significance, very little is known at the molecular, genetic and biochemical level of the genes and pathways that are responsible for the synthesis, accumulation and regulation of volatile compounds. In order to understand the biological basis of aroma biosynthesis we characterized and differentiated four stages in terms of maturity parameters, aroma-related volatile compounds, and gene expression levels. We cloned and quantified by qPCR the gene(s) encoding alcohol dehydrogenase (ADH), lipoxygenase (LOX) and pyruvate decarboxylase (PDC), key enzymes involved in alcohol and aldehyde synthesis. As fruit ripening progressed, we observed an increase in adh transcript levels simultaneously with a decrease in aldehydes (i.e. hexanal and hexenal). We think that further studies to be performed in terms of identifying and characterizing these genes in P. armeniaca will contribute to understand overall aroma development during fruit ripening (Funded by FONDECYT 1060179).



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Water transport in Grape Berries: A Functional analysis

Claudio Silva C1., Herman Silva R.1 and Gabriel Sellés V.Sch.2 1. Universidad de Chile, Facultad de Ciencias Agronómicas, Departamento de producción agrícola. 2. Instituto de Investigaciones Agropecuarias. Centro Regional de Investigación La Platina. Water transport in grape berries has been studied with several methods, nevertheless, it has not been made on live grapes on the plant. A water transport analysis in grape berries was made using electronic trunk and berry diameter sensors in grapevines cv. Crimson Seedless. It was found that the trunk growth is stopped at the veraison beginning, and an important water flor resistance fall at the phase II start of the berry development, due to the change of the phloem unloading, from a simplasmic to an apoplasmic pathway. A back flow from the berry to the shoot at night-time is proposed, wich does not cause berry shrinkage. The daily berry shringake happened because of its transpiration. Trabajo Financiado por FONDECYT, Proyecto Nº 1020837


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Sugar and fructan synthesis and superoxide dismutase activity as protection mechanisms for water deficit and heat shock stress in aloe Barbadensis miller (Aloe

vera). 1Salinas C., 1Ramírez, I., 1Huerta C., 2Sagardía S., 1Vega A., 2Silva H., 1Stange C., 1Handford M. 1Cardemil L. 1Departamento de Biología, Facultad de Ciencias, and 2Departamento de Ingeniería y Suelos, Facultad de Ciencias Agronómicas, Universidad de Chile. Aloe vera can be cultivated in desert regions of northern Chile. The objective of this research was to determine the molecular protection that Aloe vera has towards desert conditions. We investigated the increase of sugars, fructans, and protection given the presence of Heat Shock Proteins (HSP) and superoxide dismutase (SOD) towards oxidative stress induced by water stress and heat shock. The plants were watered with a volume of 8L/h/plant during 1hour, 45 min, 30 min and 15 min (T1, T2, T3 and T4 respectively). Independently, leaves from one year old plants were subjected to 25, 35 and 45ºC for two hours. Under these conditions, the number of leaves, plantlets and the gel produced was determined, and the sugars, polyfructans, and the activity of SOD were quantified, identifying isozymes of SOD. Simultaneously, primers were selected to quantify the expression of the HSP70, HSP100 and ubiquitin genes. RT-PCR amplifying the 3´end detected bands of the expected size of these HSP genes. The results show that the vegetative growth and the amount of gel were affected by T4. The sugars and fructans increased in T4 and gradually with heat shock. The total proteins decreased in the leaf bases with water deficit. In the leaf tips, proteins diminished in T4. The activity of SOD increases in the leaf bases with water deficit. Electrophoresis of the native SOD detects of 6-7 isoforms, one corresponding to Mn-SOD. One Cu/Zn-SOD isoform increases in the leaf tips under T4. We conclude that Aloe vera diminishes its growth, amount of gel and proteins in T4. The plant adjusts osmotically under these stresses, synthesizing sugars and fructans, and it is protected from oxidative stress by increasing SOD activity. (FONDECYT 1070899 and Proyecto MULT 05/30-2 U. Chile).


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Role of anthocyanin in UV-B radiation resistance in Arabidopsis mutant (eskimo1) Gallardo, J., Bascuñan-Godoy Donoso-Ñanculao, G.,, L., Bravo, L.A., Corcuera, L.J. Laboratorio de Fisiología Vegetal, Departamento de Botánica, Universidad de Concepción. The high UV-B dose application is responsable for severe damages at DNA level, proteins and cell membranes that could result in plant death. Has been reported some plants resist the UV-B radiation avoiding the damage accumulating pigments as anthocyanin capable of absorb this radiation. The mutant Arabidopsis (eskimo1) has anthocyanin connstitutive levels 10 folds greater than the wild type. Because this finding the protective role of this pigment against UV-B radiation in Arabidopsis thaliana was studied. 0.8 mW/cm2 of UV-B radiation dose was applied during 40 minutes to both mutant and wild type. This dose is 10 times greater than the UV-B radiation observed at tropic. The photoinactivation rate was 30% greater in wild type Arabidopsis than mutante ones. Five days after treatment 5 of 9 mutant plants survived (55.6%) while 0% of wild type resisted the treatment. These results suggest a correlation between greater anthocyanin content and protection against UV-B radiation in Arabidopsis. LB and JG CONICYT scholarship holders. GD MECESUP scholarship holder.


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Mechanisms of Photoprotection against low temperature-induced photoinhibition in Colobanthus quitensis

Luisa Bascuñan, Alejandro Navarrete, Luis J. Corcuera, León A. Bravo Universidad de Concepcion, Facultad de Cs. Naturales y Oceanográficas, Departamento de Botánica The combination of low temperatures and high light are propitious conditions to produce photoinhibition. However plants have developed different strategies to avoid photoinhibition and repair photodamage. Among these mechanisms the decrease in the capacity to absorb light energy has a very important role. This involves thickness of leaf parenchyma, chloroplast distribution in the cell, thylakoid membranes staking and the size of antenna complex adjustments. Colobanthus quitenis grow from Mexico to Maritime Antarctic, always associated to low temperature and high light environments, including the Andes Mountain. We studied two populations of C. quitensis that inhabits at 2700 and 3500 m.a.s.l. The parenchyma from plants growing at 3500 m.a.s.l. is formed by four layer of longer cells, than those from 2700 m.a.s.l. which have only three layer. On the other hand, plants from 3500 m.a.s.l. have more stacked thylakoids per individual granum (about 40) than chloroplasts of plants from 2700 m.a.s.l. (about 12 thylakoids per granum). The level of Lhcb2, one of the of main proteins of the antenna complex, is a 400% higher in plants from 2700 than from 3500m.a.s.l. Therefore, the decrease of probability of light absortion could be considered as an important mechanism of photoprotection at higher elevations.


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Identification of a new almt1 gene promoter allele conferring aluminum tolerance to wheat: advances on its molecular and functional characterization

Monica Mathias, Braulio Soto, Gastón Muñoz And Javier Zúñiga§

Unidad de Biotecnología de Plantas, INIA Carillanca, Casilla 58-D, Temuco, Chile.

§: corresponding author: [email protected]

Aluminum toxicity in acid soils prevailing in the central and south regions of Chile severely limits the productivity of wheat. The allelic variability at the promoter of the ALMT1 gene, which encodes an aluminum activated malate transporter, has been linked to increased levels of malate exudation and aluminum tolerance in wheat. We studied the frequency of the six previously reported ALMT1 promoter alleles in a collection of 76 wheat cultivars and landraces grown in Chile. A new allele, designated as Type VII, was discovered in the population at a frequency of 2,6%. From plants carrying this new allele, a 2,1 kb fragment containing the ALMT1 promoter region was PCR amplified, cloned and sequenced. The available sequence suggest that Type VII allele would have a structure such as the BA region, linked to higher transcriptional activity of the ALMT1 gene, would be repeated four times. The evaluation under hidroponic culture of isogenic lines carrying alleles Types I, V or VII, showed that the Type VII allele confers the highest level of aluminum tolerance. These results suggest that the Type VII allele could be used in breeding programs to develop new aluminum tolerant wheat cultivars.

Research funded by grant FIA-PI-C-2005-1-A-064. Acknowledgments:

• CONICYT • INACH



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Selection of salinity tolerant hybrid Grape Rootstocks

Azúa-Bustos A, Prehn D, Kremer C, Soto A, Manzur JP, Perez-Harvey J y Zurita A. Global problems of desertification impose an increasing demand towards finding alternatives for the adaptation of grapevines to challenging environmental conditions. Since Vitis vinifera is susceptible to salinity, its growing potential is limited in northern Chile. However, other species of the Vitis genera are much tolerant to this kind of stress. One example is Vitis champini (Ramsey, Salt Creek), a rootstock that has a high tolerance to salinity, and also shows other positive traits. In this work, a segregating population of hybrid seeds was generated through semi-controlled crosses (open pollination) with Vitis champini as pollen receptor. The total percentage of in vitro germination in the presence of growing concentrations of NaCl (25, 50, 75, 100 y 120 mM NaCl) was determined by measuring normal root development and foliar expansion. This determined the selection of a concentration of 75 mM of NaCl as a critical threshold damage value, which was further used as a selection factor during subsequent in vitro germination selection rounds. Through this screening process, a first subpopulation of individuals was obtained (8,9%, corresponding to 344 plantlets from a total of 3879 seeds) capable of germinating and developing normally in vitro in this stress condition. These plantlets were acclimated, and went a second screening process using NaCl in ex vitro conditions in order to identify the most tolerant phenotypes. In this screening, 9,5 % (33 plantlets out of 347) of the plants were highly tolerant to irrigation with 30 mM NaCl. Boron then will be used as a selection factor in ex vitro conditions in a next round of selection for identifying a subpopulation of individuals tolerant to both NaCl and Boron. The selected germplasm may be used as new rootstocks able to tolerate high salinity stress conditions.


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Antioxidant capacity of ethanolic extracts of Tristerix tetrandus

Jerez S., Zúñiga G.E. Laboratorio de Fisiología y Biotecnología Vegetal, Facultad de Química y Biología, Universidad de Santiago de Chile (USACH) Quintral, Tristerix tetrandus, is a parasitic species of the Loranthaceae family. This species is widely distributed in the center-south part of Chile, affecting populus (Poplar spp.) and maqui (Aristotelia chilensis). Studies with extracts the European parasitic species, Viscum album, show that these extracts have anticancer, hypotensive and antirheumatic properties. In the traditional medicine, Quintral is used to stabilize the circulatory system, to improve the memory, and to reduce headache. In this study the antioxidant capacity of ethanolic extracts (1:5 p/v) of leaves of T. tetrandus was evaluated. The antioxidant power of the extracts was measured by means of a test of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). As control we used chlorogenic acid. The total phenolics were assayed colorimetrically by means of the Folin-Ciocalteu method. The ID50 of chlorogenic acid (1 mg/ml) was 7.6 ul, whereas the ID50 of the quintral extract was 6.2 ul. The extracts contains a high polyphenolic content (2.6% expressed as gallic acid). High-performance liquid chromatography analysis revealed the presence of several phenolic acids, the most abundant was rutin. It is proposed that the antioxidant capacity of the Quintral extracts could be determined at least in part by its content of rutin.


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