CRP ICGEB Research Grant 2007

General Information Application and Conditions for

This Call for Research Proposals is open to ICGEB Affiliated Centres only as specified in Annex A to this Form

Programme Priorities
Research proposals must be relevant to ICGEB objectives and its potential contribution. Only projects addressing original scientific questions will be considered; among these, preference will be given to projects providing a clear indication towards practical application(s) of particular relevance for the applicants country.

Submission of Applications
Project proposals must be submitted, individually, by the Principal Investigator (PI). Applications must be completed in English, using the enclosed forms, and submitted in original with one double-sided copy duly endorsed by the ICGEB Liaison Officer (see Form A for name and full postal coordinates). Forms submitted without the necessary signatures and endorsements will not be considered. The maximum number of grants proposals that may be submitted by each country cannot exceed three (3) and Liaison Officers are requested to submit to ICGEB, on or before the closing date, a final list summarising their endorsed proposals.

Investigating groups do not qualify for renewal and/or submission of projects prior to the evaluation and satisfactory termination of the ongoing projects.

Financial Considerations
The maximum duration for any project is 36 months. The maximum annual contribution per ICGEB grant awarded cannot exceed Euro 25,000. Funds must be used in accordance with the research grant application guidelines of the enclosed application.

DEADLINES
31 March 2007: endorsement. Suggested deadline for submission to the ICGEB Liaison Officers for

ICGEB CRP Application Form 2007

30 April 2007:

Endorsed applications to be submitted to ICGEB Office of the Director-General, attn. Ms. Barbara Argenti, Padriciano 99, I-34012 Trieste, Italy. Endorsed copies may be sent via fax or e.mail ahead of the original

ICGEB CRP Application Form 2007

For ICGEB internal use only Proposal Number:

(assigned by ICGEB)

Date of receipt:

FORM A
Padriciano 99 I-34012 Trieste Italy Tel: +39-040-3757382 Fax: +39-040-3757361 E-mail: bargenti@icgeb.org Web: www.icgeb.org

CRP - ICGEB Research Grant

2007 Application Form

Project title

Comparative Genomics of Red and Black Maca Ecotypes

Principal Investigator* Institute Address:

Telephone: Fax: E-mail:

Luis Destefano-Beltran Av. Honorio Delgado San Martn de Porres Lima 31 Lima, Per +51 1 3190000 ext. 2702 +51 1 3821762 ldestefano@upch.edu.pe
Date of Submission

Signature

30-April-2007

*Name of the scientist being responsible for the coordination of research and for the submittal of the application on Forms A
and B. The Principal Investigator must be an employee of the Institution receiving the grant.

Endorsed by ICGEB Liaison Officer of PER. (Country)

Full Name

Dr. Augusto Mellado Mndez

Signature

Date of Submission

30-April-2007

ICGEB CRP Application Form 2007

FORM A.1

Confirmation by the Institute

We hereby confirm that is working in this Institute as

Luis Julio Csar Destefano-Beltrn

(Principal Investigators full name)

Associate Professor
(position)

since

01/06/2006
(dd/mm/yy)

The Principal Investigator is authorised to request the funds which will be necessary to carry out the proposed research. Should this application be selected for funding, the administrative official authorised to sign the contract on behalf of the P.I.s Institute will be:

Legal Representative* Institute Address:

Dr. Jaime Villena Chvez

(Full name)

Av. Honorio Delgado 430 San Martn de Porres Lima 31 Lima, Per Telephone: +51 1 319000 ext. 2257 Fax: +51 1 3190004 E-mail: jvillena@upch.edu.pe

Signature

Date of Submission

30-April-2007

Official stamp of the Institution

* An official of the Institution fully empowered to enter into contracting arrangements on behalf of the Institution

ICGEB CRP Application Form 2007

FORM B

100
Part I

Curriculum Vitae of Principal Investigator

(attach additional pages if necessary)

Surname First Name Birthdate (dd/mm/yy) Nationality Position title Institute address

Destefano-Beltrn Luis Julio Csar 29/08/1956 Peruvian Associate Professor Av. Honorio Delgado 430 San Martn de Porres Lima 31, Lima, Per +51 1 3190000 ext. 2702 +51 1 3821762 ldestefano@upch.edu.pe

Tel: Fax: E-mail:

Have you previously received a grant (CRP) from the ICGEB? If yes, please indicate the Ref. No.: CRP/_______________________

YES

NO

Have you previously received an ICGEB Fellowship? If yes, please indicate whether it was a

YES

NO

short-term fellowship pre-doctoral fellowship post-doctoral fellowship

Part II - Education (begin with initial professional education) Institute & location Universidad Nacional Mayor de San Marcos, Lima, Per Louisiana State University, Baton Rouge, LA, USA Degree B. Sc. Ph.D. Year 1980 1991 Field of study Biological Sciences Biochemistry

Part III Current & previous employment

ICGEB CRP Application Form 2007

06/06 Heredia, Lima, Per. 08/03 - 05/06 Laboratory, ARS, 06/02 - 04/03 Universidad de La 05/02 04/03 12/01 04/02 03/99 - 12/01 03/96 03/99 07/95 02/96 04/93 06/95 Agriculture, CIAT, 04/91 04/93 Belgium.

Associate Professor, Genomics Research Unit, Universidad Peruana Cayetano Plant Physiologist, Sugar Beet and Research Potato Unit, Northern Crop Science USDA, Fargo, North Dakota, USA. Visiting Associate Professor, Graduate Program in Plant Molecular Biology, Frontera, Temuco, Chile. Director of Research, VitroGen, SA, Temuco, Chile. Ag-Biotech Consultant, Pittsburgh, PA, USA. Assistant Director of Research, Demegen, Inc, Pittsburgh, PA, USA. Assistant Scientist, Institute of Paper Science and Technology, Atlanta, GA, USA. Postdoctoral Associate, Cornell University, Geneva, New York, USA. Senior Research Fellow, Unit of Biotechnology, International Center of Tropical Cali, Colombia. Postdoctoral Research Fellow, Laboratory of Genetics, University of Ghent, Ghent,

Part IV - Current & previous grants awarded Funding (last two years) Co-Principal Investigator, Molecular and Biochemical Characterization of the Hypoctyl Transcriptome in Maca (Lepidium meyenii) (2nd Part), 1 year (2006), $25,000, financed by CONCYTEC-PROCOM, Lima, Per. Co-Principal Investigator, Molecular and Biochemical Characterization of the Hypoctyl Transcriptome in Maca (Lepidium meyenii) (1st Part), 1 year (2005), $40,000, financed by CONCYTEC-PROCOM, Lima, Per. Part V - Publications

(Attach publication list including peer reviewed research papers, books and patents pertinent to this application for the last 5 years only)

Publications (2000 - ) Leal-Rojas, P., Gutirrez-Moraga, A., Destefano-Beltrn, L., Salvo-Garrido, A., Gidekel. M. (2007). Differential gene expression in Cala Lily Plants (Zantedeschia spp.). Agrociencia 41: 141-152. Destefano-Beltrn, L., Knauber, D., Huckle, L., Suttle, J. (2006). Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues. Plant Molecular Biology 61: 687-697. Destefano-Beltrn, L., Knauber, D., Huckle, L., Suttle, J. (2006). Chemically forced dormancy termination mimics natural dormancy progression in potato tuber meristems by reducing ABA content and modifying expression of genes involved in regulating ABA synthesis and metabolism. Journal of Experimental Botany 57: 2879-2886 (Cited by 1, Google Scholar). Gidekel, M. (*), Destefano-Beltrn L. (*), Garcia, P., Fuentes, L., Alberdi, M., Bravo, L., Corcuera, L. and Gutierrez, A. (2003). Identification and characterization of three novel coldacclimation responsive genes from the extremophile hair grass Deschampsia antarctica Desv. Extremophiles 7: 459-469 (*) First authorship. (Cited by 4, Scopus). Casas-Mollano A, Destefano-Beltrn L (2000). Characterization of a cDNA encoding a TTG1-like protein from Apple Fruits. Plant Phys. 122:1457 (Cited by 3, Google Scholar) Patents (applications filed) Rapid and efficient micro propagation system for Copihue (Lapageria rosea). Gidekel Manuel; Vallandares I; Rivas Y; Gutirrez Ana; Destefano-Beltrn Luis; and Guerra, E. Non-provisional patent application number 10/857,994. Filing date 06/01/2004. Pending.

US

Low temperature responsive nucleotide sequences and uses thereof. Gidekel, Manuel; Gutirrez, Ana, Destefano-Beltrn Luis and Leal, P. Non-provisional US patent application number 11/639,747. Filing date 12/15/2006. Pending. Transgenic plant expressing Deschampsia antarctica modified gene transcript and uses thereof. Gidekel, Manuel; Valladares, I; Leal, P; Destefano-Beltrn Luis and Guerra E. US provisional patent 60/567,125. Filed 05/02/2004. Term expired 05/02/2005.

ICGEB CRP Application Form 2007

Plant promoter regulated in response of cold temperatures. Gidekel, Manuel; Gutirrez, Ana; Destefano-Beltrn, Luis and Cuba M. US provisional patent 60/567,135. Filed 04/30/2004. Term expired 04/30/2005.

ICGEB CRP Application Form 2007

200
201

Project
Title
(To be specific within the area of research - maximum 60 characters)

Comparative Genomics of Red and Black Maca Ecotypes 202 Abstract

(Provide a summary of your research proposal)

Maca (Lepidium meyenii Walp.) is a hardy perennial plant cultivated exclusively between 3500 and 4500 m of altitude in the central Peruvian Andes. The Maca plant is a rosette of frilly leaves with an enlarged fleshy underground organ formed by the taproot and the lower part of the hypocotyl. These organs swell during growth and develop into a storage tuberous-like organ, the tuber, resembling turnip (Brassica rapa L.) or radish (Raphanus sativus L.). Maca, the only known member of the Brassicaceae family native to the Andes, has been used traditionally by Peruvians living at high altitudes as a nutrient, an energizer and fertility-enhancing properties. Due to the latter, maca is also known as the Peruvian ginseng and sold all over the world in capsules as a herbal and food supplement. In the last 5 years numerous studies have validated the fertility-enhancing and other medicinal properties present in the maca plant. Thus, it has shown to improve spermatogenesis in male rats as well as sperm count and sperm motility in normal men without affecting serum hormone levels. Also, it was shown to increase litter size in normal adult female mice. Most of the work in Maca has been concentrated to three ecotypes: Black, Yellow, and Red. Recent studies have provided experimental evidence to support that most if not all nutritional, fertility-enhancer, and anti-oxidant properties previously described for Maca are ecotype or cultivar-specific. For instance, whereas Black maca has the highest effect on sperm production, Red maca does not have any effect. On the other hand, while Red maca reduces prostate size in rats treated with Testosterone Enanthate (TE), Black maca does not. Surprisingly, Yellow Maca present both activities although at intermediate levels. In spite of these numerous studies, nothing is known about the secondary metabolites responsible for these biological effects and their biosynthetic pathways. Such knowledge is of crucial importance to by-pass the low product yield of various secondary metabolites in plants or plant cell cultures. The long-term goal of our group is to characterize the gene-to-metabolite correlations for the main Maca active compounds. In this project we want to take advantage of the biological differences between the Black and Red Maca ecotypes. As genomics has developed into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits. We plan to develop a Maca microarray to gene profile both ecotypes. Also, we will perform exploratory non-targeted metabolome analysis of both ecotypes. Finally, we will establish Black- and Red Maca-derived cell suspension cultures and develop Agrobacterium tumefaciens-mediated transformation protocols for these cultures. Our project will certainly benefit of the tremendous advances made with two other members of the same family, Arabidopsis thaliana and Brassica oleracea, in which, for example, almost all the genes encoding the enzymes involved in glucosinolate and indole alkaloids biosynthesis have been identified. Please do not exceed this space

ICGEB CRP Application Form 2007

300

Introduction
Provide a critical evaluation on the status of research in the proposed field
(Maximum 1 page)

Maca (Lepidium meyenii Walp) is the only member of the Brassicaceae family native to the Andes and grows exclusively between 3500 and 4500 m above sea level. The maca is a rosette of frilly leaves with an enlarged fleshy underground organ formed by the taproot and the lower part of the hypocotyl. These organs swell during growth and develop into a storage tuberous-like organ, the tuber, resembling turnip (Brassica rapa L.) or radish (Raphanus sativus L.). Maca is a cultivated plant and different ecotypes o cultivars have been described according to the color of its tuber ranging from White to Black. The most abundant ecotype is the Yellow Maca followed by the Red and Black Maca ecotypes. Maca is traditionally employed in the Andean region for its fertility-enhancement properties. Lately, Maca has established itself as the most important and symbolic medicinal plant from Per. Indeed, there has been a sustained increase in the demand for maca in Japan, Europe and the U.S., the often called maca boom (Li et al., 2001). In the last several years numerous studies have validated the fertility-enhancing properties and other medicinal properties present in the maca plant. Gustavo Gonzles and his group have described several physiological effects present in Maca extracts. The first evidence that maca improved spermatogenesis was reported in male rats (Gonzles et al., 2001a). Thereafter, Gonzles et al., (2001b) demonstrated that maca also improved sperm count and sperm motility in normal men without affecting serum hormone levels. Moreover, it was shown that Yellow Maca restored spermatogenesis in models where spermatogenesis was diminished. For instance, oral administration of aqueous extracts of Yellow Maca prevented disruption of spermatogenesis in rats exposed to high altitude (Gonzles et al., 2004) and the deleterious effect of a single dose of the organophosphate insecticide Malathion on spermatogenesis in mice (Bustos-Obregn et al., 2005). Also, Yellow Maca extracts reversed the lead acetate induced damage on reproductive function in male rats (Rubio et al., 2006) and increased litter size in normal adult female mice (Ruiz-Luna et al., 2005). Recently, it has been established that Maca has different biological effects according to its ecotype. From the three ecotypes studied (Gonzles et al., 2006) Black Maca showed the best reproductive effect. In fact, treatment for 42 days with Black Maca resulted in higher Daily Sperm Production (DSP)/testis, higher DSP/g, higher epididymal sperm count, and higher epididymal sperm motility in male rats. Yellow Maca had an intermediate effect, whereas Red Maca had no effect at all on these parameters. However, this is not the only biological difference between Maca ecotypes since in other study Red Maca reduced prostate size in normal rats preventing its increase in Testosterone Enanthate(TE)-treated rats (Gonzles et al., 2005). Unpublished results from their laboratory seem to indicate a dose-dependent reduction in prostate weight with increasing amounts of red maca benzylglucosinolates as well as an apparent positive effect of altitude at which these plants are grown with their biological and medicinal properties. In other studies, Valentov et al., (2006) have demonstrated that both aqueous and methanolic maca extracts have estrogenic activity comparable with that of silymarin in MCF-7 cell line. Additionally, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. In contrast, Yu et al. (2006) found an anti-senility effect in mice present in ethanolic maca extracts by improving free radical metabolism and enhancing immune function. In spite of these numerous studies, nothing is known about the secondary metabolites responsible for these biological effects and their biosynthetic pathways. On the other hand, such knowledge is of crucial importance to by-pass the low product yield of various secondary metabolites in plants or plant cell cultures (Goossens et al., 2003). Functional genomic approaches are powerful molecular tools to accelerate comprehensive investigations of cellular metabolism in specialized tissues or whole organisms. Plant secondary metabolism has been studied using comparative quantitative trait loci mapping, 2D gel electrophoresis-based proteomics, or transcript analysis tools, such as differential display, EST databases, and microarrays. Now it is possible to determine gene-to-metabolite correlation through the comprehensive analysis of gene expression (transcriptomics) and metabolite accumulation (metabolomics) (Aharoni et al., 2000; Guterman et al., 2002; Goossens et al., 2003; Mercke et al., 2004; Gachon et al., 2005; Hirai et al., 2005; Tohge et al., 2005). Our group (Luis Destefano-Beltrn & Magadalena Pavlich) has recently initiated a project to characterize the maca tuber transcriptome. To date, we have constructed a cDNA substractive library using Yellow Maca tuber mRNA as tester and mRNA from the rest of the plant as driver and random sequenced ca. 5,000 clones. Most of the partial cDNAs show high levels of sequence homology (>80%) to Arabidopsis thaliana and Brassica sp genes. Currently, we are in the process of obtaining the full-length cDNAs of several tuber-specific clones since all the cDNA fragments are rather small due to the cDNA substractive technique. The long-term goal of our groups (LDB & MP and GG) is to characterize the gene-to-metabolite correlations for the main active compounds in both Red and Black Maca. We think that an in-depth understading of the main Maca biosynthetic pathways, as in other systems, will enable the exploration of metabolic engineering as a potential effective approach to increase the yield of specific metabolites by enhancing rate-limiting steps or by blocking competitive pathways. In this project we want to take advantage of the remarakable biological differences between Black and Red Maca ecotypes as they offer an interesting model of how putative few gene differences could lead to dramatic biological properties. As Genomics has developed into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to
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ICGEB CRP Application Form 2007

elucidate the molecular basis of complex traits. We plan to develop a Maca microarray to gene profile both ecotypes. Also, we will perform exploratory non-targeted metabolome analysis of both ecotypes. Finally, we will establish Black- and Red Maca-derived cell suspension cultures.

10

ICGEB CRP Application Form 2007

400
401

Research Project
Define specific research activities to be pursued during the project period and provide a comprehensive description of the techniques to be used and the advantages of the suggested methodological approach. Please include any selected relevant references.
(Maximum 5 pages, including references)

The purpose of this project is the use of comparative genomics to study the transcriptomes of Red and Black Maca ecotypes. Our long term interest is to dissect the main biosynthetic and metabolic pathways in Maca and by doing so improving and expanding the use of Maca as an experimental model for the study of peruvian medicinal plants. Also, this is the first attempt to lay the ground for a more aggressive value-capture in the Maca plant the biochemical and genetic information stored in its genome. In other words, the molecular identity and the biosynthetic pathway of the active compounds. Such in-depth knowledge, when available, could enable, for example, the development of specialized and efficient cell suspension lines dedicated to the production of specific compounds or through genetic modification produce novel compounds. Consequently, we envision, at the end of this three-year project, an increase in the intellectual property derived from Maca for the benefit of Universidad Peruana Cayetano Heredias technological portfolio and Pers interests. Finally, we propose to perform an exploratory look at the metabolomes of these two contrasting Maca ecotypes using non-targeted metabolome analysis. The specific research activities to be developed during this three-year project are: 1.- Comparative microarray analysis of Black and Red Maca Ecotypes. Rational The expression activities of all genes represented in an organisms genome at any given time or at any given tissue or organ constitute a complex phenoytpe that is closely connected with, but not only dependent upon, the genotype. Actually, gene expression profiles represent the primary level of integration between environmental factors and the genome, providing the basis for differential protein synthesis which in the end results in complex phenotypes. Consequently, by comparing gene expression profiles of different strains, populations or even species, one can directly study the molecular basis of phenotypic variation (Renn et al., 2004). We are particularly interested in identifying molecular components of Red and Black Maca biological properties and propose to use comparative microarray analysis of their tubers transcriptomes to identify them. We will construct a Maca microarray, the MACA-Chip. Random unique cDNAs from Red and Black Maca tuber normalized cDNA libraries will be used to prepare this cDNA microarray. cDNA libraries rich in clones containing complete coding sequences with 5-and 3-untranslated regions can be used to obtain entire sequence information for each transcript in a single cloning step, which is invaluable for high-throughput transcriptome analysis. However, one obstacle is the differential abundance of various transcripts. Usually, 10-20 abundant genes account for at least 20% of the cellular mRNA mass, several hundred genes of medium abundance comprise 40-60% of the mRNA mass, and several thousand rare genes may account for 20-40% of the mRNA mass. Normalization of cDNA libraries decrease the prevalence of clones representing abundant transcripts, so that relative transcript concentrations are equalized to a considerable extent. Normalized cDNA libraries are best to accelerate EST projects, transcriptome analysis, functional screening and rare gene discovery (Zhulidov et al., 2004). Tuber normalized cDNA library preparation and generation of EST collection mRNA from Black and Red Maca tubers will be separately used to prepare normalized cDNA using the Super SMART PCR cDNA synthesis kit (Clontech) and the TRIMMER cDNA normalization kit (Evrogen) essentially as described by Zhulidov et al., (2004). Each normalized cDNA will be cloned into the pGEM-T-easy vector (Promega). 5000 randomly picked colonies from each library will be sequenced by the Genome Sequencing Center (GSC) at Washington University in St. Louis, MO, USA. Plating, DNA purification and sequencing have been quoted at a rate of ca. 0.45 Euros per clone. Sequence analysis and annotaion of this EST collection will be done in Per. Microarray preparation and analysis MACA-Chip will be constructed by printing PCR amplified cDNA of selected clones to represent as many unique transcripts as possible for both Maca ecotypes. Printing of glass slides will be done in GSC, St. Louis, USA. mRNA isolated from Red and Black Maca tubers will be reverse transcribed to generate Cy3 and Cy5 fluorescent labeled first strand cDNA probes. cDNA probes will be
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ICGEB CRP Application Form 2007

competitively hybridized to the MACA-Chip. mRNA will be isolated in Peru and sent to the Microarray Core Facility of the GSC in St. Louis, USA where cDNA labeling, microarray probing and data analysis will be performed. Two technical replicates will be performed on each of two Maca samples for a total of 4 hybridizations. Also, Cy3 and Cy5 dyes will be swapped between mRNA of the two ecotypes for an additional technical replicate, such that Black Maca cDNA will be labeled at least once in green (Cy3) and once in red (Cy5) to avoid gene-by-dye effects. Genes will be considered expressed if the hybridization intensity is fourfold or greater than the average hybridization intensity for the internal controls in one or both channels. Genes will be considered differentially expressed if the difference in normalized hybridization intensities between cDNAs derived from Black or Red Maca is greater than twofold after subtracting the standard error on all spots (5 Hybridizations/Data Analysis have been quoted at a rate of 760 Euros each for a total of 3,800 Euros). Verification of Expression Profiles by Northerns/ RT PCR RNA gel-blot (Northerns) or RT-PCR analysis will be performed on selected differential expressed genes to confirm the microarray data. We expect to characterize several up- or down-regulated genes specific to each Maca ecotype. In any case, standard protocols from the literature will be followed. Program of Activities and Relevance Maca tubers are harvested from May to July when they are at their maximum size, about 5 cm in diameter. In the first year, we expect to isolate mRNA from both Maca ecotypes, Black and Red, and use it to prepare two normalized tuber cDNA libraries. Sequencing of 5,000 random picked colonies from each library is expected to be completed by the end of the first year as well as the analysis of the sequences and their annotation. Maca microarray preparation and analysis will be done in the second year. Validation of differential expressed cDNA clones will be done in the second half of the second year. In the third year, a subset (or all) of these genes will be characterized in greater detail according to standard protocols: Maca full-length cDNAs will be isolated, if necessary, comprehensive tissue-specific and developmental expression analyses will be performed. It is assumed that genes differentially expressed in Red Maca will somehow be involved with prostate reduction whereas genes preferentially expressed in Black Maca will be implicated in sperm increase production and other related physiological properties. 2.- Non-targeted metabolome analysis of Red and Black Maca (exploratory). Rational The next challenge in refining the search for biosynthetic genes is to make a parallel analysis of transcript and metabolite profiles. Significant correlations between the metabolic contents and the expression of relevant genes have been demonstrated (Guterman et al., 2002; Urbanczyk-Wochniak et al., 2003; Mercke et al., 2004; Hirai et al., 2005). For non-targeted metabolome analysis, it is necessary to combine several analytical technologies, particularly those based on mass spectrometry such as gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry (LC-MS), and Fourier-transform ion-cyclotron mass spectrometry (FT-MS). Thus, primary metabolites are analyzed by GC-MS while secondary metabolites are analyzed by LC-MS. Previously, Ganzera et al., (2002) used reversed phase HPLC to chemical profile maca methanolic extracts from commercial maca products but no indication was given as to the maca ecotype. We do not intend to perform a comprehensive metabolome analysis such that the molecular identity of each component is determined. Our only goal for this project is to establish the extent of the differences of the Red and Black Maca metabolomes. Do they differ only in a handful of components? Are their metabolomes significantly different? Plant Material For unbiased non-target metabolome analysis it is necessary to have at least six samples of the same origin. Consequently, whole tubers of each ecotype will be homogenized, divided in six portions and extracts prepared in exactly the same manner for all of them. Aqueous, ethanolic and methanolic extracts will be prepared from at least six samples from each ecotype. Metabolome Analysis Maca metabolome analysis will be outsourced to the Metabolomics-Core facility of the University of California in Davis, CA, USA and analyzed by LC-MS. The aqueous, ethanolic and methanolic extracts of Black and Red maca will be analyzed. Analysis for all 36 samples have been quoted in 1800 Euros. Pairwise comparisons will be made for each type of extract. Initially, we will only concentrate on
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ICGEB CRP Application Form 2007

those compounds present in one ecotype but absent in the other ecotype. In case these compounds are unknown further work will be needed which will include MS and NMR studies. Since chemical identification is much more complex, expensive and time-consuming additional funding will be secured elsewhere. Program of Activities Preferentially we plan this activity for the second year pending yearly budget allocation for this project. 3.Establishment of Cell Suspensions Cultures for Red and Black Maca

Rational In the last several years there has been a considerable interest in investigating the possibility of exploiting plant cell cultures as an alternative source of secondary metabolites products. Also, plant cells cultures provide an excellent system for studying biosynthesis of secondary metabolites. The biochemical nature of the secondary metabolites involved in the ecotype-specific properties of Black and Red Maca are unknown so there is a need to have an experimental system to exploit the vast amount of information generated by transcriptome analysis. A tyypical molecular tool to dissect gene function include candidate gene overexpression and gene knockout via RNAi technology. As a first step, we will develop stable cell suspension cultures from both Maca ecotypes. Protocol Methods for the establishment of cell suspension cultures are well documented in the literature (ie. Franklin and Dixon, 1994; Razdan, 2003). Initially, tuber explants will be isolated, cleared from contaminants and placed and maintained in a suitable media to induce callus induction. The ratio and composition of growth regulators will be determined empirically. A good starting point could be media used for the establishment of callus in other Brassicaceae. Once established, callus cultures will be transferred to the same media without the agar. Growth curves and subculturing schedules will be determined empirically. Initial explants could be tuber sections of maca plants germinated in vitro or tuber disk-sections obtained from freshly harvested maca hypoctyls. Since callus cultures contain mixtures of cells that are not uniform in size, shape, metabolism and most importantly in our case, pigmentation, an effort will be done to establish uniform pigmented cell lines Validation of Ecotype-specific properties If cell suspension cultures are established successfully they will be evaluated for the presence or absence of their native biological properties. To do this (aqueous, methanolic or ethanolic) extraction protocols will be developed for secondary metabolite isolation which will be assayed for retention of their parents biological properties. These tests will be peformed in Dr. Gustavo Gonzles lab at UPCH. Program of Activities We expect to have the first cell suspension cultures established by the end of the first year. The biological assessment of their biological properties will be made in the second year. In the third year, the transcriptome and metabolome of the validated cell lines will be analyzed pending on budget resources. Briefly, gene expression of cell suspension cultures retaining their original biological properties will be compared to that of their original tubers and/or to the other ecotypes cell lines. Similar design will be followed for the analysis of the cell suspensions metabolome. 4.- Development of Stable Transformation Protocols of Red and Black Maca Cell Suspensions Cultures. Rational Standard Agrobacterium-mediated transformation, starting with roots, leaves, cotyledons or other explants, require laborious procedures and, above all, well established and routine protocols to regenerate normal plants from those same explants. In the event that a Maca cell suspension line retains the biological properties of its original ecotype, Agrobacterium-mediated transformation could become a powerful tool to dissect gene function through the use of the proper constructs (RNAi, constitutive, etc). Protocol
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ICGEB CRP Application Form 2007

A starting point will be to follow the technique described by An (1985) for tobacco suspension culture. Agrobacterium tumefaciens LBA4404 carrying the pBI121 plasmid or a derivative (containing the Gus reporter and the proper selector genes) will be used. Points to be adjusted empirically are co-incubation time, amount of Agrobacterium and antibiotic concentration in the plating media. After co-cultivation, cells will be washed several times with culture media containing cefotaxime to remove Agrobacterium. Cells will be plated on cell culture media supplemented with cefotaxime and the right antibiotic. Plates will be stored at 25 C in the dark until calli formation is observed, usually 2 or 3 weeks. Calli exhibiting high GUS activities will be pressed through a steel sieve to obtain very small cell agregates and then further diluted in cell culture medium. Transgenic cells will be plated at very low densities and repeated twice to eliminate residual bacteria nesting within the calli.

Program of activities Since only cell lines which retain the biological properties of their parent ecotype will be used, this activity will be performed in the third year or earlier. References Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J. et al., 2000. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12: 647-661. An, G. 1985. High efficiency transformation of cultured tobacco cells. Plant Physiol. 79: 568-570. Bressan, R.A., Zhang, C., Zhang, H., Hasegawa, P.M., Bohnert, H.J., Zhu, J.-K. 2001. Learning from the Arabidopsis experience. The next gene search paradigm. Plant Physiol 127: 13541360 Bustos-Obregn, E., Yucra, S., Gonzles, G.F. 2005. Lepidium meyenii (Maca) reduces spermatogenic damage induced by a single dose of malathion in mice. Asian J. Androl 7: 71-76. Clough, S.J., Bent, A.F. 1998. Floral Dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735-743. Curtis, I.S., Nam, H.G. 2001. Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floraldip method plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res. 10: 363-371. Franklin , C.I., Dixon, R.A. (1994). Initiation and maintenance of callus and cell suspension cultures. In Plant Cell Culture: A Practical Approach. Dixon, R.A., Gonzales, R.A. Eds. 2 Ed. IRL Press, N.Y. Gachon, C.M.M., Langlois-Meurinne, M., Henry, Y., Saindrenan, P. 2005. Transcriptional co-regulation of secondary metabolism enzymes in Arabidopsis: functional and evolutionary implications. Plant Mol. Biol. 58: 229-245. Ganzera, M., Zhao, J., Muhammad, I., Khan, I.A. 2002. Chemical profiling and standardization of Lepidium meyenii (Maca) by Reversed Phase High Performance Liquid Chromatography. Chem. Pharm. Bull. 50: 988-991. Gonzles, G.F., Ruiz, A., Gonzles, C., Villegas, L., Crdova, A. 2001a. Effect of Lepidium meyenii (Maca) roots on spermatogenesis of male rats. Asian J. Androl. 3: 231-233. Gonzles, G.F., Crdova, A., Gonzles, C., Chung, A., Vega, K., Villena, A. 2001b. Lepidium meyenii (Maca) improved semen parameters in adult men. Asian J. Androl. 3: 301-303. Gonzles, G.F., Gasco, M., Crdova, A., Chung, A., Rubio, J., Villegas, L. 2004. Effect of Lepidium meyenii (Maca) on spermatogenesis in male rats acutely exposed to high altitude (4340 m). Journal of Endocrinology 180: 87-95. Gonzles, G.F., Miranda, S., Nieto, J., Fernndez, G., Yucra, S., Rubio, J., Yi, P., Gasco, M. 2005. Red maca (Lepidium meyenii) reduced prostate size in rats. Reproductive Biology and Endocrinology 3: 5.

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ICGEB CRP Application Form 2007

Gonzles, C., Rubio, J., Gasco, M., Nieto, J., Yucra, S., Gonzles, G.F. 2006. Effect of short-term and long-term treatments with three ecotypes of Lepidium meyenii (Maca) on spermatogenesis in rats. Journal of Ethnopharmacology 103: 448-454. Goossens, A., Hkkinen, S.T., Laakso, I., Seppnen-Laakso, T., Biondi, S., De Sutter, V., Lammertyn, F., Nuutila, A.M., Sderlund, H., Zabeau, M., Inz, D., Oksman-Caldentey, K-M. 2003. A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc. Natl. Acad. Sci. USA 100: 8595-8600. Guterman, I., Shalit, M., Menda, N., et al. 2002. Rose scent: genomics approach to discovering novel floral fragrance-related genes. Plant Cell 14: 2325-2338. Hirai, M.Y., Klein, M., Fujikawa, Y., Yano, M., Goodenowe, D.B., Yamazaki, Y., Kanaya, S., Nakamura, Y., Kitayama, M., Suzuki, H., Sakurai, N., Shibata, D., Tokuhisa, J., Reichelt, M., Gershenzon, J., Papenbrock, J., Saito, K. 2005. Elucidation of Gene-to-Gene and Metabolite-to-Gene networks in Arabidopsis by integration of Metabolomics and Transcriptomics. J. Biol. Chem. 280: 25590-25595. Horvath, D.P., Schaffer, R., West, M., Wisman, E. 2003. Arabidopsis microarrays identify conserved and differentially expressed genes involved in shoot growth and development from distantly related plant species. Plant J. 34: 125-134. Li, G., Ammermann, U., Quirs, C.F. 2001. Glucosinolate contents in Maca (Lepidium peruvianum Chacn) seeds, sprouts, mature plants and several derived commercial products. Economic Botany 55: 255-262. Mercke, P., Kappers, I.F., Verstappen, F.W.A., Vorst, O., Dicke, M., Bouwmeester, H.J. 2004. Combined transcript and metabolite analysis reveals genes involved in spider mint induced volatile formation in cucumber plants. Plant Physiol. 135: 2012-2024. Quirs, C.F., Epperson, A., Hu, J., Holle, M. 1996. Physiological studies and determination of chromosome number in Maca, Lepidium meyenii (Brassicaceae). Economic Botany 50: 216-223. Razdan, M.K. 2003. Introduction to Plant Tissue Culture. 2nd Ed. Science Publishers, Inc., Enfield, NH, USA. Renn, S.C.P., Aubin-Horth, N., Hofmann, H.A. 2004. Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray. BMC Genomics 5: 42. Rubio, J., Riqueros, M.I., Gasco, M., Yucra, S., Miranda, S., Gonzles, G.F. 2006. Lepidium meyenii (maca) reversed the lead acetate induced-damage on reproductive function in male rats. Food and Chemical Toxicology (doi: 10.1016/j.fct.2006.01.007). Ruiz-Luna, A.C., Salazar, S., Aspajo, N.J., Rubio, J., Gasco, M., Gonzles, G.F. 2005. Lepidium meyenii (Maca) increases litter size in normal adult female mice. Reproductive Biology and Endocrinology 3: 16. Taji, T., Seki, M., Satou, M., Sakurai, T., Kobayashi, M., Ishiyama, K., Narusaka, Y., Narusaka, M., Zhu, J.-K., Shinozaki, K. 2004. Comparative genomics in salt tolerance between Arabidopsis and Arabidopsis-related halophyte salt cress using Arabidopsis microarray. Plant Physiol. 135: 1-13. Tohge, T., Nishiyama, Y., Hirai, M.Y., Yano, M., Nakajima, J., Awazuhara, M., Inohue, E., Takahashi, H., Goodenowe, D.B., Kitayama, M., Noji, M., Yamazaki, M., Saito, K. 2005. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 42: 218-235. Trieu, A.T., Burleigh, S. H., Kardailsky, I. V., Maldonado-Mendoza, I. E., Versaw, W. K., Blaylock, L. A., Shin, H., Chiou, T.-J., Katagi, H., Dewbre, G.R., Weigel, D., Harrison, M. J. 2000. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 22: 531-541.

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ICGEB CRP Application Form 2007

Urbanczyk-Wochniak, E., Luedemann, A., Kopka, J., Selbig, J., Roessner-Tunali, U., Willmitzer, L., Fernie, A.R. 2003. Parallel analysis of transcript and metabolic profiles: a new approach in systems biology. EMBO J. 4: 1-5. Valentov, K., Buckiov, D., Kren, V., Peknicov, J., Ulrichov, J., imnek, V. 2006. The in vitro biological activity of Lepidium meyenii extracts. Cell Biology and Toxicology 22: 91-99. Yu, L.-J., Zhang, Y.-Z., Jin, W.-W., Ji, Z.-Y., Xiong, W.-T. 2006. Anti-senility effect of ethanol extract in rhizome of Lepidium meyenii in mice. Chinese Traditional and Herbal Drugs 37: 81-83. Zhulidov, P.A., Bogdanova, E.A., Shcheglov, A.S., Vagner, L.L., Khaspekov, G.L., Kozhemyako, V.B., Matz, M.V., Moroz, L.L., Lukyanov, S.A., Shagin, D.A. 2004. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 32: e37.

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ICGEB CRP Application Form 2007

402

Potential for training of young scientists

(Specify if training of young scientists and any travel are foreseen. Please indicate the receiving laboratory and purpose of the visit)

Unlike University labs in developed countries, peruvian university labs can not afford to hire permanent technical assistance (ie. Research Assistant or Associates). Consequently, there is great potential for training of young scientists starting with undergraduate students as they are expected to prepare a thesis as part of the requirements for their graduation. Also, the College of Sciences and Phylosophy at Universidad Peruana Cayetano Heredia, UPCH, hosts a Master and a Doctoral program in Biochemistry and Molecular Biology so there is also great potential for graduate students training. Dr. Luis Destefano-Beltrn joined the Genomics Research Unit at UPCH on June 1 st of 2006. His research interests include functional genomics of medicinal plants, fruit ripening of tropical fruits and potato molecular physiology. In the last 2 years and with funding from CONCYTEC (Peruvian NSF) he has started to characterize the Maca tuber transcriptome in collaboration with Dr. Magdalena Pavlich (Laboratory of Plant Tissue Culture, UPCH). He is currently supervising a doctoral student who is carrying most of the work. If this grant is approved he will be able to attract other graduate students from UPCH graduate programs. Dr. Destefano-Beltrn is the recipient of a repatriation fellowship from UPCH and for the next 2 years he is expected to develop an innovative program in plant molecular biology starting from scratch. Also, he is expected to train Ph.D. and M.Sc graduate students. Travel is mainly anticipated by the end of the second and/or third year to present results at scientific meetings by PI and graduate students. However, some unplanned travel at the moment this proposal is written is possible for the first year in the form of workshops or courses (CHSL Courses, Gordon Conferences, etc) relevant to this project.

403

Facilities available in the Investigating Teams laboratory

(Provide a detailed list of the infrastructure and equipment available and necessary for the proposed research)

This is a partial list of the common equipment available in the building Laboratorios de Investigacin y Desarrollo, LID at UPCH. I am supposed to get my own funding to equip my lab at the Genomics Research Unit in the LID building. Common equipment for the LID Building: A Beckman Scintillation Counter, a Perkin Elmer Gas Chromatograph, a Perkin Elmer Atomic Absorption spectrometer, a Perkin Elmer espectrophotometer, a Beckman Avanti-J25 refrigerated centrifuge, a L2-75B Beckman Ultracentrifuge, two ultralow freezer 70C, a Biorad Molecular Imager System, Water purification systems: Two Millipore reverse osmosis system and two Millipore Mili Q Plus system, two ice machines, two Barnstead/Thermoline sterilizing ovens, two cold rooms and a -18 C room. One Hemco, two Labconco and two Fisher Hamilton Fume Hoods. A Mettler analytical balance and a Metler top loading balance. Two autoclaves, Sterilmatic and Yamato. A Biorad Benchmark Microplate Reader, a Virtis Freeze dryer, a New Brunswick environmental incubator shaker. While I get my own equipment, I have access to the equipment of the Molecular Biology Unit (MBU): Equipment available at the MBU housed in the LID Building: thermal cyclers machines (2), electrophoresis tanks for vertical and horizontal gels, gel dryers, manual DNA sequence apparatus, CHEF apparatus, power supply units, refrigerated centrifuge (1), RT centrifuge, Micro-centrifuges, Ultra deep freezer, Freezers, Refrigerators, Water baths,Iincubators, Gel Documentation System, laminar flow cabinet, CO2 incubators, Liquid N2 tank, Microscopes. Equipment available at the Genomics Research Unit (RGU): Thermal Cyclers MJ Research/Biorad (2), Constant Power Supply 220V 50Hz Series II, Refrigerated Centrifuge Hermle MK2, Mega-Gel Dual HighThroughput Vertical Electrophoresis Unit, VWR Water Baths Model 1211 (6L), Laboratory Refrigerator/ Freezer combination, (2) Submarine Horizontal Gel kit 20 x 40 cm, Transiluminator UV Spectroline Fisher, Economy Stirring Hotplates general purpose Fisher, Imperial Standard Incubator Barnstead/Lab-Line, VWR Ultra Low Temperature Upright Freezer, Kodak Gel Logic Imaging Systems, Sony UPD895 printer for imaging system, Eppendorf electroporator 2510.

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ICGEB CRP Application Form 2007

500 Financial Contribution requested to ICGEB (all figures to be indicated in Euro)

To carry out the research of the approved projects, the ICGEB will enter into a formal contract with the Institution to which the Principal Investigator belongs. ICGEB does not fund salaries and infrastructural support, e.g. maintenance and rental of capital equipment, buildings, etc. The ICGEB funding will cover the part of research to be carried out in the laboratory of the Principal Investigator only. Please provide annual phasing of the requested funds.

1st year Equipment1 Consumables & Training2 Travel3 Literature4 Miscellaneous5 Sub total 7,500 14,000 1,000 1,250 1,250

2nd year 7,500 12,500 2,500 1,250 1,250

3rd year 7,500 12,500 2,500 1,250 1,250

Total per budget category 22,500 39,000 6,000 3,750 3,750

25,000

25,000

25,000

75,000

TOTAL CONTRIBUTION REQUESTED TO ICGEB

75,000

1Equipment
This budget category must not exceed 30% of the total grant requested/awarded and is to be spent preferably during the first year of the project, for the purchase of basic standard laboratory equipment and/or components that are necessary to the implementation of the research activities which will be carried out with the ICGEB grant. Items not funded by ICGEB: Purchase of major equipment (i.e., equipment costing more than Euro 10,000), office furniture or fittings, computer hardware or software. Small pieces of equipment of the value of Euro 500 or less (e.g., micropipettes) are to be considered as consumables and charged to this budget line accordingly. Item(s) First Year Second Year Third Year Thermal Cycler 3,750 x 2 3,750 MiniCentrifuge 3,750 Benchtop Platform Shaker 2,250 UV-Crooslinker 1,500 Dual Hybridization Incubator 3,750 Total 7,500 7,500 7,500

2Consumables & Training

This budget category is intended to cover the purchase of consumable items and short-term training visits of scientists directly involved in the project for hands-on training related to the project as well as stipends for long-term trainees from other ICGEB Member Countries (e.g., not nationals of the country to which the ICGEB grant has been awarded) operating in the project. Costs not funded by ICGEB: Salary support of Principal Investigator. Item(s) First Year Pipettes (complete set) 1,000 Horizontal Minigel Systems (different gel 300 size, x3) RNA extraction chemicals 500 PolyA-Tract mRNA Isolation Systems 500 Super SMART cDNA Synthesis kit 1,200 TRIMMER cDNA normalization kit 900 PCR Cloning Kits 500 Taq Polymerase 500 Microarray Analysis (outsourced) Metabolome Analysis (outsourced) Sequencing (outsourced) 4,500 Chemicals (various) 1,000 Glassware 1,000 Plasticaware 2,000 Others 100 Total 14,000 Second Year 300 1,000 3,800 1,800 1,500 500 2,000 1,600 12,500 Third Year -

2,000

3,200 500 3,000 3,800 12,500

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ICGEB CRP Application Form 2007

3Travel
This budget category must not exceed 10% of the total grant requested/awarded and is intended to cover the participation of scientists directly involved in the project in major conferences/meetings related to the project objectives. Description of foreseen expenditures Travel for short term visit Travel to Major Conference Total First Year 1,000 1,000 Second Year 500 2,000 2,500 Third Year 500 2,000 2,500

4Literature
This budget category must not exceed 5% of the total grant requested/awarded. Funds may be used to cover the purchasing costs of scientific literature and reference material (e.g., books, subscription to journals and periodicals) and/or publication costs of scientific manuscripts pertinent to the project in peer-reviewed journals (e.g., page charges and publication fees). Description of foreseen expenditures Books, journals Publication costs Total First Year 1,250 1,250 Second Year 1,250 1,250 Third Year 500 750 1,250

5Miscellaneous
This budget category must not exceed 5% of the total grant requested/awarded and is intended for communication expenses (e.g., telephone, fax, Internet connection, postal and courier services) and other contingencies that are considered essential for the project implementation (e.g., photocopy, stationery material, etc.). Costs not funded by ICGEB: normal administrative and overhead expenses of the Institution. Description of foreseen expenditures Telephone and Fax Postal and Courier Services Photocopy Lab Notebooks, others Total First Year 200 750 100 200 1,250 Second Year 200 750 100 200 1,250 Third Year 200 500 100 450 1,250

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ICGEB CRP Application Form 2007

600

Proposed Referees
(Provide the name and full coordinates of a maximum of 3 referees who would be willing to review your proposal. Please note that the ICGEB will have the sole responsibility in deciding whether or not a proposal will be submitted for evaluation to the referee(s) listed below)

Referee No. 1

Surname First Name Institute address

Valerio, Jr. Luis Office of Food Additive Safety Division Biotechnology & GRAS Notice Review, USA

Tel: Fax: E-mail:

Office: (301) 436-1253 luis.valerio@fda.hhs.gov

Referee No. 2 Surname First Name Institute address

Shilin Chen Institute of Development of Medicinal Plants, Beijing, China Director, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, P. R. China No. 151 Malianwa North Road, Haidian District, Beijing 100094, P. R. China POSTCODE100094

Tel: Fax: E-mail:

slchen@implad.ac.cn

Referee No. 3 Surname First Name Institute address

Tel: Fax: E-mail:

Flores, Hector E. Dean and Professor of Biological Sciences, 201 Centennial Hall College of Science 601 University Dr., Texas State University San Marcos, TX 78666, USA 512-245-2040 hf12@txstate.edu

ANNEX A - ICGEB Member States with an Affiliated Centre

eligible to apply for funding under the CRP ICGEB Research Grant Programme

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ICGEB CRP Application Form 2007

CRP - ICGEB Research Grant Application Form 2007

Check List for Principal Investigator

Have you completed all the sections of this application form in English?

X X X X X

Have you signed Form A and obtained the endorsement of the ICGEB Liaison Officer of your country (refer to Annex A for name and full postal coordinates)? Has the Legal Representative of your Institute signed Form A1?

Have you completed the section 500 (e.g., Financial contribution requested to ICGEB) according to the application guidelines ? Is the budget expressed in Euro?

Please submit one original with one double-sided copy, not bound

For ICGEB Liaison Officers Please note that incomplete applications will not be processed

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