ANNUAL REPORT FOR AWARD NSF# 9813360
Genomics of Plant Stress Tolerance
Hans J Bohnert; U of Arizona

Participant Individuals:

    Co-Principal Investigator(s) Ray A Bressan; Paul M Hasegawa; David W Galbraith; Jian-Kang Zhu

    Senior personnel(s) Eduardo Misawa; Lanying Zhang

    Post-doc(s) Robert Fischer; Shinji Kawasaki; Wujun Ma; Joon-Hyung Kim; Qinmiao Sun; Patricia Ayoubi; Rosanna Muralla; Kap-Hoon Han; Chun-Peng Song; Marcela Nouzova; Shin Kore-eda; Zhizhong Gong; Tracie Matsumoto; Albino Maggio; Hisashi Koiwa; Gyung-Hye Huh; Michael Deyholos

    Graduate student(s) Lisa David; Quiolan Wan; Brad Postier; Hua Jun; Haihui Huang; Shuhua Yuan; Anamika Ray; Jiana Zhu; Byeong-ha Lee; Harish Rekapally; Brian O'Dell; Ryan T Lyle; Thomas Rendon; Hanz Richter; Ravi Thambusamy; Wei Yan; Jing Wang

    Undergraduate student(s) Susan Brazille; Lorenzo Caracciolo; Miriam Eafon; Heather Ferrea; Luke Knipe; Gabriel Scara; Gontran Zepeda; Julianne Lazarr; Junag-Hong Chong; Tema Starkman; Ryan Kylin; Lori Impson; David Bellovin; Monica Dennis; Fadi Alkayal; Amy Huesby; Tonja Pappan; David Pesta; Amanda Hedges; Suzan Ro; Allisson Rank; Davina Bufford; Chien-Kuen Woo; Jeremiah Cox; Brian Rhodes; Ivan Blanco; Saul Leite; Jeson Martdjaja; Sarah Hopping

    Technician, programmer(s) Tristan Dow; Christine B Michalowski; Becky Stevenson; Georgina Lambert; Mary Ann F Cushman; Roushan Samad; Summers Wilson

    Technical school faculty(s): Jing Wang

    Other -- specify(s): Rolf A Prade; John C Cushman; Robert L Burnap; Jaqueline Yale; Yuko Tanaka; Elwira Slinwinska; Mika Nomura; Casey Lu; Shuji Yokoi

Partner Organizations:

Oklahoma State University Agricultural Experiment Station: Financial Support; In-kind Support; Facilities; Collaborative Research; Personnel Exchanges

The OSU Agricultural Experiment Station had promised to return its share of IDC back to the project investigator (amounts to $70,694 and will be paid out over four years) for personnel support and equipment purchases.

Dr. Cushman was also recently awarded a competitive, internal grant by the OAES for the purchase of equipment (e.g., Humidity chamber/dust cover) and software (e.g., CloneTracker, GeneVision) to enhance our microarray construction and analysis efforts. Total award = $17,280).

Oklahoma State University: Financial Support; In-kind Support; Facilities; Collaborative Research; Personnel Exchanges

OSU Arts & Sciences committed 15% cost-share for both Prade and Burnap. A mutually agreed decision was reached that all available first year monies would be used to renovate Pradeís lab and subsequently Burnapís allocation would become available. Things are on track in this regard. Burnap will be receiving the funds in 3 installments over the course of FY2000, FY2001, and FY2002 (see attached letter). This money will be utilized for equipment, array costs, and for salaries including converting a technician position to a second postdoctoral associate.

As of May 12, 1999 the Associate Dean for A&S Research has fully funded his promise to contribute with 15% of the total amount of the NSF subcontracted award as indicated in the attached letter.

Purdue University: Financial Support; In-kind Support; Facilities; Collaborative Research; Personnel Exchanges

All commitments made by the Dept. Horticulture and Landscape Architecture, and the School of Agriculture has been fulfilled.

University of Arizona Agricultural Experiment Station: Financial Support; In-kind Support; Facilities; Collaborative Research; Personnel Exchanges

Promised ó salary for postdoc and part of a technician and equipment; all has been provided by UA.

We have ordered PCR machines, computers, shakers, incubators, etc, with matching fund from the College of Agriculture. CoA is building a room-sized walk-in growth chamber, also with matching fund.

Other collaborators:

    Dr. Jose Pardo, Univ. Valencia, Spain (long-time collaborator with Drs. Bressan & Hasegawa) Dr.Omar Pantoja, UNAM, Cuernavaca, Mexico, collaboration Bohnert group Dr. Howard Griffiths and collaborators, U. Newcastle, UK, collaboration Bohnert and Cushman groups various other collaborators, including discussions with Dr. T. Wilkins (UC Davis), Pam Green (MSU), Virginia Walbot (Stanford), Julian Schroeder (UCSD), Wolf Frommer (Univ. Tuebingen, Gemrany), Arun Majumder (Bose Institute, Calcutta, India) Yasuyuki Yamada and several of his collaborators (NAIST, Nara, Japan), John Bennett (IIRI, Manila, Philippines), John O'Toole (Rockefeller Foundation, Bangkog, Thailand), Arjula Reddy (Univ. Hajderabad, India), Andrew Smith (Plant Sciences Oxford, UK), Klaus Winter (STRI, Balboa, Panama), Ulrich Heber (Wuerzbyurg, Germany), Ulrich Luettge (Darmstadt, Germany), Thomas Rausch (Heidelberg Germany), Toshiyuki Fukuhara (Tokyo, Japan) and this list is not complete.

Activities and Findings:

Research Activities:

I. EST projects

The focus for the first year was to establish most or all cDNA libraries for the project and to begin EST analysis. We are nearly there (see the list below) with the cDNA libraries. In addition, we have approximately 4,000 new sequences (i.e., in addition to those of the Cushman group). This number will double by the end of year 1.

A. Libraries from Stressed Plant/Algae/Fungal Models We have so many different libraries that we developed a common nomenclature to all ESTs: First two letters: AA to ZZ ó denotes organism, organ/tissue/cells, developmental stage, stress level, duration of treatment. AA to AZ for Arabidopsis thaliana (Columbia; Lehle Seeds) DA to DZ for Dunaliella salina MA to MZ for Mesembryanthemum crystallinum (Klaus Winter collection, STRI, Panama, 1990) NA to NZ for Nicotiana tabacum SR1 (from MPI Cologne, 1983) OA to OZ for Oryza sativa (Nipponbare, Pokkali; John Bennett, IRRI, 1994) ZA to ZZ for Zea mays (B73, Vicky Chandler, UA, 1998).

The following libraries have been obtained as of June 1, 1999:

Mesembryanthemum crystallinum libraries (UA & OSU)

MA- primary leaves, plants 5-6 weeks of age, grown in hydroponics (0.5 Hoagland's), no stress, harvest at daytime; cloned into lambda- UniZap. (seqs. ~1,200)

MB ó primary leaves, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 30 h stress, cloned in lambda UniZap (seqs. ~2,200)

MC ó roots; plants 5-6 weeks of age, grown in hydroponics (0.5 Hoagland's), no stress, harvest at daytime, cloned in Bluescript SK+ (no sequences yet)

MD ó roots, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 1h stress, cloned in Bluescript SK+ (no sequences yet)

ME ó roots, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's); 6 hours stress, in Bluescript SK+ and (older one) in lambda UniZap (several plates in progress, 3 plates done)

MF ó roots, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 12h stress, Bluescript SK+ (several plates in progress, 3 plates done)

MG ó roots; plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 30h stress, lambda UniZap and Bluescript SK+ (several plates in progress)

MH ó roots, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 78h stress, Bluescript SK+ (no sequences yet)

MI ó seedlings, 14 days-old, 3d stress at 250 mM NaCl, Bluescript SK+ (4 plates in progress)

MJ ó secondary leaves , 8-9 weeks-old, 5 d, 500 mM, Bluescript SK+ (no sequences yet)

MK - primary leaves, 6 weeks old; 6-7 d stress at 500 mM NaCl, Bluescript SK+ (no sequences yet)

ML ó flowers & developing seedpods, >12 weeks of age, 6 weeks in 500 mM NaCl, Bluescripts SK+, (3 plates done, 3 in progress)

MM ó epidermal bladder cells, >12 weeks, 6 weeks in 500 mM NaCl, Bluescript SK+ (5 plates done, 3 in progress)

MN ó side shoots, 6 weeks, 3 d 500 mM Nacl, Bluescript SK+ (no sequences yet)

MO ó apical meristems and leaf primordia, 5 weeks-old, no stress, Bluescript SK+ (5 plates sequenced or in progress)

MP ó apical meristems and leaf primordia, 6 weeks-old, 3 d 500 mM NaCl, Bluescript SK+, (3 plates done, 12 plates in progress)

MQ ó library with plant material as in library MH, for yeast complementation [pYES2].

MR ó normalized primary leaves, plants 5-6 weeks of age, grown in hydroponics (0.5 Hoagland's), no stress, harvest at daytime; cloned into Bluescript SK+ (no sequences yet)

MS ónormalized primary leaves, plants 5-6 weeks of age, grown in hydroponics, stress 400 mM NaCl (in 0.5 Hoagland's), 48h stress, cloned in lambda UniZapXR (no sequences yet)

MT óprimary leaves, plants 5-6 weeks of age, grown in hydroponics, drought 6h stress, cloned in lambda UniZapXR (no sequences yet)

MU ónormalized primary leaves, plants 5-6 weeks of age, grown in hydroponics, drought 6h stress, cloned in lambda UniZapXR (no sequences yet)

Arabidopsis thaliana (Columbia) libraries (UA & Purdue)

AA ó leaves, flowering plants, 12 weeks old, 20 h, 200 mM NaCl, Bluescript SK+ (1 plate done, several plates in progress)

AB ó leaves, seedlings, 2-3 weeks-old, combined after 3, 6, 9, 12h stress, 200 mM NaCl, Bluescript SK+, (1 plate done, several plates in progress)

AC ó seedlings, leaf & root , 2-3 weeks, 20 h stress , 150 mM NaCl, Bluescript SK+ (several plates in progress)

AD ó 14 d seedlings, 4h 100 mM NaCl, in yeast expression vector; (no sequences yet)

AE ó 14d-seedlings, 2-4h stress, 160 mM NaCl(no sequences yet)

AF ó roots, flowering plants, hydroponics (0.25 x Hoagland's), 6 h stress, 200 mM NaCl (no sequences yet).

Oryza sativa (Nipponbare) libraries (UA)

OA ó root, 3-4 weeks old, 19nh stress, 200 mM NaCl Bluescript SK+ (1 plate done, several plates in progress) OB ó leaf, 3-4 weeks-old, 19 h stress, 200 mM NaCl, Bluescript SK+ (no sequences yet)

Oryza sativa (Pokkali) (UA) (libraries done for OC,OD.OE, no sequences yet)

OC ó 1 week-old seedlings, roots, no stress,

OD ó 1 week-old seedlings, root, 1D 150 mM NaCl

OE ó 1 week-old seedlings, root, 2-3d stress 150 mM NaCl

OF ó 2 weeks-old, whole plant, 1 w stress 150 mM NaCl ó (library under construction).
Zea mays (B-73) libraries (UA)

ZA ó roots, 2 weeks-old, 24 h stress, 150 mM NaCl, Bluescript SK+, Sequences by Stanford consortium, deposited in GenBank in part

ZB ó leaves/shoots, 2 weeks old, 24 h stress, 150 mM NaCl, Bluescript SK+. Sequences by Stanford consortium, deposited in GenBank in part. Sequencing by this group (~1,000 sequences), see: http://www.zmdb.iastate.edu/

Nicotiana tabacum (SR1) libraries (no funds for sequencing ESTs) (UA)

NA ó root & leaves, 4 weeks-old, a mixture of stress: 6, 24, 30, 48 h stress, 200 mM NaCl, UniZap XR & Bluescript SK+

NB ó leaves, mature plants, no stress, UniZap XR & Bluescript SK+

Dunaliella salina (Purdue)

DA ó up-shift 0.5 M NaCl to 3 M NaCl (will be in yeast expression vector) (in progress; sequencing in year 2)

B. EST Sequencing Progress:

The following EST sequences have been completed:
 

Organism

June 1, 1999

October 1, 1999 (projected)

A. thaliana

500

3,000

M. crystallinum

3,000

6.000

O. sativa 

300

2,000

Z. mays 

1,000

1,000

D. salina

0

0

A. nidulans

12,485

15,000

II. Microarrays

A. Yeast microarray analysis

The yeast microarray analysis (plus/minus NaCl) is completed, a manuscript will be submitted soon. The abstract is included in the finding section. Tables, which provide these data, are being readied for web-release. In addition, we are preparing lists for genes that are regulated in yeast under salt stress and plant homologues that have been observed in the EST sequencing project. These amount to 64 transcripts at present.

B. Mesembryanthemum microarrays

Robotic instrumentation for the construction (e.g., Gene Machines, Inc. Omnigrid and Cartesian Technologies, Inc. PixSys Systems) and analysis (GSI Lumonics, Inc. ScanArray® 3000 MicroArray Biochip Scanning System) of cDNA microarrays has been purchased and now fully operational at both UA and OSU. We have successfully completed the printing and analysis of arrays containing 400 elements. Cushman lab (OSU) has begun to array Mesembryanthemum leaf-derived DNA elements will complete the printing and analysis of first generation microarrays containing 1536 elements consisting of ice plant cDNA inserts by the end of June, 1999. These will be made available to any interested laboratory. At present, the approximately 4,000 EST sequences established (in addition to the ESTs established at OSU) are being prepared for microarray analysis by people in the Galbraith and Bohnert labs. Bohnert lab is focussing on arrays of transcripts from M. crystallinum roots, and from specific tissues (flower/ epidermis/ etc.). More development work on the microarrays is being done in the Galbraith lab (in collaboration with Dr. Deyholos) to test hybridization conditions, amount of DNA printed and printing of slides with elements that represent different abundance classes of transcripts. We are also assembling microarrays for different pathways and categories ó chaperones, mitochondria or chloroplast functions, and biochemical pathways. The utility of microarrays is not nearly well enough known and previously reported data sets seem to require new interpretations. The printing and analysis is being done with relatively small arrays of several 100 elements.

C. Synechocystis microarrays

The Burnap lab has developed algorithms and databases to directly program large, ordered sets of oligonucleotides in 96-well format by deconvoluting the multi-step path going from pairs of 96 well microtiter dishes containing sets of gene-specific forward and reverse PCR primers to an organized layout of gene fragments on a microarray slide. No commercial programs are available for this operation (note that Arrayer programs such as Cartesianís 'Clone-Tracker' will not do this). We are in the process of connecting the modules together and integrating it with automated primer design and error checking functions. These latter two have been performed semi-manually to generate the first 450 primer pairs for the phase I array. Spotting the Phase I arrays containing 450 open reading frames will commence in June 1999. Approximately 70 Synechocystis genes homologous to higher plant and yeast salt stress genes known or predicted from the plant stress genomics consortia or defined by other researchers as being involved in salt stress tolerance have been identified. Approximately 900 primers (450 genes) for the Phase I arrays have been designed using genomic sequence data and web-based design programs. This is a subset of the genome containing all identified Synechocystis photosynthesis and respiration genes, as well as a large number of known stress and regulatory genes. Phase II arrays will contain the full genome and are planned for early next year. Synechocystis RNA isolation protocols have been optimized and are known to be suitable for reverse transcription methods used to generate two color CyDye probe for microarray expression profiling. Note that there are presently no published procedures for this in prokaryotes.

D. Aspergillus microarrays

The A. nidulans EST/clone collections present a significant tracking problem (that translates into wrongfully assigned clone names to sequences deposited in GenBank). To circumvent the inconsistency between clone name and DNA sequence information, the Prade lab decided to print PCR products produced with primers that uniquely amplify each ORF rendering a uniform (~500 bp) gene probe preferentially located at the 3í-end of the open reading frame. We have developed a Web-based (Intranet available only) primer production resource that uses Web-primer software (Stanford University) and a series of CGI programs that manage and check the accuracy of primers and PCR amplification products. To date over 2600 primer pairs have been designed and primers are being synthesized on a 96 well Mermade system (Bruce Roe, University of Oklahoma). Preliminary and test slides will contain 96, 288 and >4,000 genes respectively.

E. Arabidopsis and Dunaliella Microarrays

EST collections for Arabidopsis and Dunaliella microarrays are under development.

F. Other Microarraying Projects

Galbraith lab is also involved in NSF-supported maize gene discovery grant (V. Walbot, Stanford University, and PI). Details of this work are reported under that project. However, close synergistic links are maintained between the personnel working on the two different projects. The NIH-funded Medical Oncology Program Project (MOPP) grant is competing for renewal this year. A key component of the renewal proposal is the inclusion of microarraying as a core research activity at the Arizona Cancer Center. Galbraith is participating in this proposal as a co-P.I. Further, over the last 18 months, he has worked closely with the AZCC scientists to establish large (5,000 element) microarrays comprising human cDNAs and ESTs. These activities complement those required for microarraying in the stress project, and include methods for array element production, RNA signal amplification, and the evaluation of different surfaces for printing, arrayer software debugging, and data analysis and archiving. Galbraith also serves as co-P.I. with Rene Feyereisen and (formerly) Ken Feldmann on two projects aimed at characterization of the cytochrome P450 gene superfamily of Arabidopsis thaliana. These projects are funded by USDA and the Human Frontiers Program, respectively. Students in his lab are working on analyzing gene expression of the different P450 family members using microarrays.

III. NEST and GFP Reporter Analysis

Dr. Chun-Peng Song has been primarily responsible for the application of NEST (nuclear expressed sequence tag) technology (Macas et al., 1998), for the analysis of genes that are up-and down-regulated in response to salt stress. His experimental strategy in brief is as follows: tobacco plants are grown hydroponically. Selected plants are subjected to salt stress for 24 h by transfer into hydroponic solutions containing 250 mM NaCl. Treated and control plants are harvested and leaf and root nuclei separately isolated by flow sorting following staining of homogenates using propidium iodide. The nuclei are lysed, and the nuclear RNA subjected to reverse transcription in the presence of magnetic beads covalently carrying oligo-dT. Following second strand synthesis, 3í cDNA tags attached to the beads are produced through treatment with NlaI. A single class of linkers is ligated onto the tags and subsets of these are sequentially amplified using PCR. The tags are displayed on polyacrylamide gels and visualized by silver staining. Comparison of lanes corresponding to treated and controls plants allows identification of transcripts whose abundance is altered as a consequence of salt-stress, as well as a comparison of effects that are tissue type-specific. Dr. Song has identified >50 transcripts of this type. The bands corresponding to these transcripts have been excised and the cDNA tags re-amplified for sequencing. BLAST analysis is underway.

Mr. Shuhua Yuan has designed and expressed GFP-based reporter constructs to track the transcriptional regulation of specific stress-induced genes in transgenic Arabidopsis plants. In particular, he is examining the regulatory 5í-upstream sequences of two stress-related aquaporin genes (MIPA and MIPB) and one root-specific ATPase gene. Constructs have been made, and transgenic Arabidopsis plants are being produced and analyzed for expression using confocal microscopy.

Mr. Bellovib has placed the NLS-GFP-GUS construct under the control of the rd29 promoter, so that NEST analysis can be done on cells responding to activation of this promoter.

IV. Insertional Mutagenesis

A. Synecchocystis

One of the major reasons for developing a PCR-mediated mutagenesis technique is the versatility that it gives to engineering chromosomes. This method provides an alternative to cloning-base procedures depending upon restriction sites. Although most of our efforts thus far have been focused upon the microarrays, some progress has been achieved in this area. The synthesis of PCR-mediated DNA fragments consisting of a marker cassette flanked by short (100 bp) homology regions to the target locus. Alternative strategies for generating marker cassettes with longer flank are ongoing. Future efforts are aimed at refining these methods and improving the transformation efficiencies.

B. Yeast

Mutations in yeast were generated originally by EMS mutagenesis resulting in the isolation of 11 osmotic sensitive mutants . Currently, the Tn3 insertion mutagenesis system is being used to isolate additional salt sensitive mutants. This system contains a lacZ reporter fusion for identification also of stress responsive promoters. Gene isolation is facilitated by plasmid rescue.

Knockout lines for selected functionally unknown ORFs - with homology to unknown plant ESTs of unknown function - are in progress.

C. Arabidopsis

Activation T-DNA (bialaphos resistance marker and 4X 35S enhancer) tagged populations are being generated by in planta transformation with Agrobacterium. The genetic background of the parental plants is RD29A::LUC (stress responsive promoter (osmotic, cold, ABA) fused to the luciferase reporter gene) or sos3, for screening suppressors of this mutant. Currently, efforts are being made to obtain a parental genotype that is expressing an desiccation-specific MIP promoter-LUC fusion for enhancer tagging. To date, about 35,000 T-DNA tagged lines in the RD29A-LUC genetic background have been produced. Mutants both with altered salt sensitivity and RD29A signaling are being isolated. To date, 200 putative mutants with altered salt responses have been isolated. Currently, co-inheritance of the T-DNA with the phenotype is being evaluated. The phenotype of 3 of the genotypes has been confirmed in the progeny. Evidence indicates that one salt sensitive genotype has an insertion near a gene encoding a LRR protein. In addition, efforts have been initiated to produce DNA pools of the T-DNA tagged population for reverse genetic identification of mutants.

We have optimized and streamlined the process of large-scale activation tagging mutagenesis in the RD29A-LUC Arabidopsis. To date, approximately 20,000 insertion lines have been generated. Out of these, 2,000 lines have been screened for stress gene regulation mutants using our luminescence imaging system. Several putative mutants were recovered and are being tested further. To prepare for future cloning of the mutations, we have tested and optimized conditions for TAIL-PCR to clone plant sequences flanking T-DNA insertions.

V. Osmotically Regulated Salt Tolerance Determinants (and Confirmation by Loss-of-Function Molecular Genetics, Salt Tolerance Sufficiency or Suppression of Stress Sensitive Mutants)

Tolerance determinants are being isolated based on differential expression in response to salt treatment. Specifically, these are being identified by: analysis of yeast genomic microarrays, subtraction of wild-type (Columbia) and sos3 (salt-overly-sensitive) Arabidopsis cDNAs (determined by high output sequencing of subtracted cDNAs), and sequencing of cDNAs that are obtained after salt treatment of Arabidopsis and Dunaliella. Na+/H+ antiporter encoding cDNAs of the glycophyte Arabidopsis and the halophyte Atriplex nummularia have been isolated. These antiporters have sequence similarity to NHE-type exchangers (yeast NHX1). Genetic analysis indicates that AtNHE functions in the tonoplast, contributing to vacuolar compartmentation of Na+. Activated yeast CaN, SPK1, Na+-ATPase encoded by ENA1, g-glutamyl kinase/g-glutamyl phosphate reductase encoded by tomPRO2, 3í(2í),5í-bisphophate nucleotidase and inositol polyphosphate 1-phosphatase encoded by SAL1 (and antisense), TOB175, and the Na+/H+ antiporter encoded by AtNHE1 are being evaluated as plant salt tolerance determinants. Both wild-type and SOS mutant Arabidopsis plants have been transformed with constructs for constitutive expression of these genes and determination of salt stress tolerance sufficiency or complementation, respectively.

VI. Bioinformatics

The main goal for the bioinformatics component of this project, during its first year, is to implement basic infrastructure for support of this project. The main focus tasks are: - purchase and install dedicated Unix server for this project - develop automated DNA sequence processing systems - develop team collaboration tools - setup a website for distribution of information generated by this project - training of undergraduate and graduate students on development and maintenance of software for genomic research

During this period, two low-end SUN workstations were upgraded to be used as development platform for this project. A Sun Ultra 60 with large capacity has been ordered and is pending delivery. 3 PCs running X-windows clients have been ordered, since they are more cost-effective than X-terminals. The PC's will also be more useful for this project since they can used for debugging of platform independent software.

Some key software has been implemented. The Phred/Cross-match/Phrap suite as well as BLAST program has been installed. An automated system updates the databases used by BLAST, so that the local version of BLAST stays identical to BLAST available at NCBI.

An automated DNA sequence processing system has been developed. The system can be used either through a web interface or command line commands in Unix shell session. Raw sequence files can be uploaded through FTP session in the user workspace. The sequences can be either manually processed through Phred/Phrap suite or BLAST. It is also possible to run through Phred/Cross-Match/Phrap and the output automatically BLASTed and the result deposited into a database. The system is in 'alpha' testing phase and will be made available to all project members as 'beta' testing phase before the end of June of 1999.

Project collaboration tools are also been implemented. Private resources - closed to general public and only accessible to team members through username and password - have been developed. Current resources provide private discussion groups involving either the whole research group or specific sub-groups, which allow focused asynchronous discussions relevant to the project. Private web sites and ftp sites were also set for distribution of information within each group or all the team members in this project.

A public web site has been developed and it is available at: The objective of this site is to distribute the information generated in this project to the open research community.

Research Findings:

I. EST Analyses

A. Mesembryanthemum crystallinum

To date, we have sequenced and analyzed 2500 ESTs from M. crystallinum leaf cDNA libraries. Another 3000 sequence templates from root, meristem, and leaf derived cDNA libraries have been prepared and are in the process of being sequenced. A semi-automated system for EST sequence analysis is now in place and being used to identify the non-redundant set of EST clones (unigene set) and full-length clones from contig assemblies. This unigene set is being used to build microarrays containing non-redundant cDNA inserts (see below). The EST database for M. crystallinum has been established in collaboration with Prade/Misawa groups. Analysis of this larger EST collection confirms our earlier assessment of the utility of stressed ice plants as a rich resource for the isolation of unknown or novel gene important to stress adaptation processes. In well-watered plants, only 17% of ESTs were unknown with another 7.8% being novel. In contrast, salt stressed plants, 30% of ESTs were unknown and 9.5% were novel. From this analysis we have identified more than 300 new potential candidates for further study by the functional genomics program. This total does not include about 100 additional genes for which functional information exists only in the form of expression data.

B. Aspergillus nidulans

12,485 cDNAs (ESTs) have been sequenced from A. nidulans. Approximately 990 (9.1%) of them have been classified in at least one of the following stress group: heat-, cold-shock, ubiquitination, detoxification, starvation or oxygen deprivation (Ayoubi P and Prade R, unpublished results). Within the stress transcripts we identified (BLAST high scoring pairs above 100 or expectation values below 10-5) 24 heat-shock genes including HSP30, HSP78, HSP104, HSP70, HSP101, HSP40 chaperones and other related polypeptides, 19 ubiquitin components, 7 vacuolar pumps and 51 general transporters (Ayoubi P and Prade RA, unpublished results). In addition, we found specific stress-related homologs such as: HOL1, a major facilitator (S. cerevisiae); HOG1, the mitogen-activated and osmosensing protein kinase (S. cerevisiae); one osmotic-growth-protein, OSM1 (S. cerevisiae); three oxidative stress resistance proteins (Zn/Cd), ZRC1 (S. cerevisiae); three low-temperature and salt responsive proteins, LTI6A (A. thaliana); one stress induced oxidoreductase - induced by heat shock, salt stress, oxidative stress, glucose limitation and oxygen limitation, GSP39 (B. subtilis); one cooper homeostasis factor (A. thaliana); three superoxide dismutases (C. albicans, A. thaliana, H. sapiens); and two catalases (P. putida, A. fumigatus). Thus, analysis of a partial EST gene complement ó 11,000 ESTs identify about 3,700 unique A. nidulans ORFs, 45% of the predicted 8,100 genes (Kupfer et. al. 1997), reveals a fairly complete picture of the KNOWN gene functions whose role have been implicated with stress and degenerative functions in other organisms.

C. Other species

We have begun sequencing of ESTs for transcripts from Arabidopsis and Oryza stressed libraries. By the end of June, we will have about 1,000 ESTs, each which will then be used for normalizing the library population before sequencing additional clones. The number of sequences depends on the availability of operations monies.

II. Microarrays

A. Yeast Genomic Microarrays

The recent elucidation of the Saccharomyces cerevisiase genomic sequence, and the availability of DNA microarrays have augmented approaches by which salt tolerance determinants can be identified, including both components of stress signaling cascades as well as effectors that are regulated by the pathways. The microarray analysis of wild type yeast (plus/minus NaCl) is completed. A manuscript will be submitted soon. The abstract is included:

Patterns of gene expression following salinity stress [1 M NaCl for 10 and 90 min., respectively] were determined using Saccharomyces cerevisiae genomic microarrays. While most regulated transcripts could be placed into functional categories, approximately 262 regulated transcripts represented functionally unknown ORFs. Early increases, 2 to 40-fold, characterized 125 ORFs and 364 transcripts increased late. ORFs down regulated, 2 to 10-fold, following osmotic stress amounted to 204. Strong increases (10-min. response) identified ORFs for enzymes involved in energy production, and amino acid/nucleotide metabolism, while the most dramatic decreases in transcript levels occurred for ORFs related to transcription. Long-term salt stress led to increases in four categories: (1) stress and defense reactions, (2) detoxification and oxygen radical stress responses, (3) membrane transporters for many different functions, and (4) energy provision and metabolism of carbohydrates and lipids. Most extensive down-regulation in late gene expression was observed for ORFs related to ribosome/protein synthesis and nucleotide metabolism. A comparison (top 300 regulated transcripts) between salt stress, radical oxygen stress and heat shock indicated a significant overlap between radical stress and salt stress, but little overlap of these with heat shock. Only 12 genes were induced by all stresses.

Tables, which provide these data, are being readied for web-release. In addition, we are preparing lists for genes that are regulated in yeast under salt stress and plant homologues that have been observed in the EST sequencing project. These amount to 64 transcripts at present.

The Purdue groups have compared differences in steady-state RNA levels of isogenic strains of salt sensitive yeast mutants, cnb1, hog1, or spk1 (mutations in different salt stress signal pathways) and wild type cells after imposition of salt stress. The scientific premise is that dissection of genes regulated by these significant salt tolerance signal cascades will add substantially to the database of determinants that mediate adaptation. These include 42, 88, and 12 mRNAs regulated specifically by the CaN, hog1, and spk1 pathways, respectively. Confirmation of the microarray data will be based on northern analysis and loss-of-function experiments.

III. NEST Analysis

Dr. Song from Galbraith's lab has analyzed 33 of 190 possible primer combinations and has found 39 bands that are upregulated by salt-stress in leaf, 27 bands that are upregulated by salt stress in root, and 2 that are down regulated in both roots and leaves. He has amplified 10 of the first category and five of the second. He aims to amplify all of them and to do the other primer combinations. Three have been sequenced so far and are being BLASTed.

IV. Insertional Mutagenesis

A. Synechocystis

Using the PCR-mediated targeted gene replacement strategy, the peroxiredoxin gene has been knockout resulting a salt sensitive phenotype, but the efficiencies of this process are low. Future efforts are focused on improving the efficiency of this process and expanding the scale of functional determinations.

B. Yeast

Molecular genetic analysis of these genotypes indicates that, besides the calcineurin (CaN) signal pathway, two others are involved in the regulation of ion homeostasis in yeast. The mutant nls2 can be suppressed by activated CaN, and serine-threonine kinase (SPK1) suppresses either CaN null or nls2 mutations, and spk1 is salt sensitive. The double mutants (spk1 and CaN null, or spk1 or nls2) are more salt sensitive than any individual mutant. Suppressors of nls2 or CaN null mutants include CIN5 (encodes leucine zipper super-family transcription factor, SCP1/2 (C-2 domain protein), and SPK1 (see attached tables).

C. Arabidopsis

The more than 20,000 tagged lines (May 99) will be the end of summer have become more than 40,000 lines. Multiplied seed populations in batches will be available by fall. Since the lines include a promoter-luciferase reporter construct, the screening for mutants with altered responses to various environmental stresses is ongoing. Several dozen lines have been found which are putatively mutated in the signaling functions leading to promoter induction.

Research Training

The project is designed to provide the following contributions to the research and teaching skills and experience of those who have worked on the project. A list provided elsewhere indicates the large numbers of faculty, postdoctoral students, graduate, and undergraduate students that we are currently training in the molecular biology and genetics of plant stress responses.

1. College faculty. The project primarily demands of faculty that they integrate their individual research programs towards pursuing a common goal, in this case that of understanding, at the molecular level, the response of plants to abiotic stress. This runs counter to the traditional model of biology research, that of the individual investigator. It is however a required demand, since the scope of the project vastly exceeds the capabilities of just one person or laboratory. Integration, moreover has to be successfully achieved at two levels, between and within different universities. Such interaction is beginning to work among the PIs and a few combinations of people, However, many persons (at all levels) are quite defensive of their turf and need to be convinced of the benefits of sharing information. This is made more complicated due to the many nationalities (from different cultures) included in the program, and due to the complexity of the sub-projects. Regular meetings of all participants within the three universities are employed to unite the various labs of that institution. These meetings also lead to multidisciplinary training and research activities, for example so that students work in more than one laboratory. Between university interactions are done (i) in the form of regular meetings of the contributing PIs and senior research associates (the first, two-day meeting was held in Tucson in February 99, and the second is scheduled for the day following the ASPP annual meeting in Baltimore), (ii) via electronic communication, primarily email. The project is clearly a learning experience for the PIs because interaction is essential for our success. It has been a steep learning curve and the interactions are not yet smooth and free of friction and misunderstanding. The project also demands that faculty recruit excellent postdocs, graduate and undergraduate students, and technical support persons. Whereas it is relatively straightforward to find qualified postdocs, identifying U.S. citizens or residents remains problematic. OSU labs have faced delays in hiring the best available post-docs due to competition from more prestigious institutions. Identification of qualified graduate students is also problematic, being a chronic situation across the U.S. Novel strategies for grad student recruitment represent a major commitment for the program in the upcoming year. At the undergraduate level, it is not at all difficult to find excellent motivated students. For example, at the University of Arizona, we have the (NSF) award-winning Undergraduate Biology Research Program (UBRP) which coordinates and funds (50% level) undergraduates to perform research in faculty laboratories. During the current year, 225 faculty are identified as sponsors in this program, 140 students being enrolled in biology laboratories across the campus. Since the program was initiated, a total of about 850 students have been supported, or which 54% are females. Under-represented minorities comprise 18%, and Asian-Americans another 12%.

2. Post-docs. The purpose of the program is to provide post-docs with a thorough understanding of the conceptual basis behind the integrated study of stress biology, to provide them access to the latest in experimental techniques and state-of-the-art instrumentation, so that they are equipped to elucidate the regulation of the genes and the gene products involved in stress responses, and to provide a nurturing and critical environment within which they can develop and hone their research skills. We aim to provide opportunities for postdocs to prepare proposals for local as well as national funding, as long as this does not disrupt their research productivity. Ultimately, we wish to prepare postdocs to be independent researchers, whether in the academic, public, or private sector, so we aim to educate them in the skills needed to be flexible, innovative and thorough researchers. In terms of teaching, the program provides opportunities for the postdocs to participate in instructional activities primarily within the research laboratory setting. We anticipate that they will be able, in the future, to participate in more formal pedagogical offerings that will be associated with the program. The postdocs also have to the opportunity to learn how to mentor graduate students through supervising their day-to-day research activities. We also aim to provide training in laboratory ethics and biosafety. Ethics training is done largely through personal example and discussions. Biosafety training has now been institutionalized.

As examples of postdoctoral training involvement in teaching, at the University of Arizona postdoctoral research associate Michael Deyholos (Galbraith lab), and postdoc Betsy Pierson (supported by the Maize Gene Discovery grant to V. Walbot, also in Galbraith lab) are organizing workshops on microarraying (see section 5). In the Bohnert lab, Dr. Robert Fischer is involved in undergraduate training at a regular basis and in introducing techniques to a group of technicians and undergraduate students.

3. Graduate students. The primary training of graduate students within the Plant Stress program depends on whether the students are pursuing Ph.D. or MS programs. For Ph.D. students, the elements of training parallel those for postdoctoral associates, but of course are at a more junior level. Students are expected to develop research skills including the ability to critically review the literature, to define important research questions, to identify the most appropriate way to address these questions, to design and carefully carry out experiments to test hypotheses, to clearly and coherently report the results obtained, and to draw conclusions. Manuscript preparation and submission skills are also emphasized.

For MS students, the primary emphasis is on developing the technical research skills needed for entry into the biotechnology industry. This requires considerable flexibility in the use of different experimental techniques and approaches, as well as social skills in the ability to work successfully in large groups.

For both types of students, participation in regular laboratory and group meetings allows development of critical thinking skills and the ability to present data in a coherent, concise, and logical manner.

4. Undergraduate students The primary purpose of our training of undergraduates is to familiarize them with the way in which biological research is pursued. This starts with the most basic of scientific and social concepts, such as how to define research questions, how to plan and perform experiments, how to record and report data, how to recognize and deal with ethical and safety questions, and how interact with other workers in the laboratory setting. The student has also to be made aware of the creative process and assisted in developing research ideas. This involves the development of critical thinking and analytical skills. At the same time, the students need both to develop technical skills and to develop a broad-based awareness of the interdisciplinary approaches needed to progress in biological research. We insist that undergraduates participate in all organized meetings of the various groups, and encourage them to present data as this develops. In terms of the research goals of the project, the major training mission for undergraduate students is to introduce them to laboratory research in the molecular genetics of plant stress and to allow them to gain valuable technical skills in a highly stimulating and productive research environment. As mentioned previously, recruitment of excellent undergraduates is not viewed as a problem at this time. The large numbers of undergraduates being trained by the stress consortium, more than 50, is a testament to our training capabilities. This large number also points to the important contribution that undergraduates are making to this research effort. As an example, the Bohnert lab is currently employing 15 undergraduate students this summer. All students, and including the four graduate students in the lab, assemble every Friday afternoon during the three summer months for discussions, which cover aspects of their work (most work in the genomics project), and the overall goals and objectives of the functional genomics project. Five of the students will remain in the fall to work on their senior theses, and two have expressed their intention to enroll into the MS program.

5. K-12 teachers and outreach to high-schools Involvement of K-12 teachers and the development of an outreach program will be a focus of our second year of activities. At the University of Arizona, we are in close contact with Ms. Carol Bender, who directs the U.B.R.P. (see above). Several mechanisms currently exist for outreach to K-12. The first, entitled the 'Science Connection' is a volunteer, student-initiated program of student placement in High School science classes. Started in 1991, this program is designed to introduce molecular biology techniques to the classroom environment. The second is aimed at a similar goal, but focuses on the teachers, through a MS program in Biotechnology offered over the summer months. This program includes laboratory training for the teachers, but also funds a recently appointed coordinator (Dr. Erin Pekol) who provides follow-up at the various schools, to ensure the programs are implemented in the classroom. Given the particular relevance of plant stress biotechnology to the desert southwest, we propose to set up formal interfaces with this program. In this respect, Dr. Bohnert is at the discussion stage in the use of mutants and mutant screening in the classrooms of teachers at St. Gregoryís High School in Tucson, and of faculty at Whitman College, Washington. Dr Bohnert is also initiating a supplemental grant request for K12 teacher training in genomics. This is in collaboration with John Fray (University of Massachusetts) who provides the perspective of an African American. Entitled: 'Genomics of Plant Stress Tolerance: Didactic Tools for the Integration of Education and Research', the proposal builds on the currently supported grant to determine how crop plants respond to stresses in their physical environment. It proposes to train K-9 teachers to employ rote functional analysis for the examination of some of the large numbers of gene knockouts and random-insertion mutants in Arabidopsis, yeast, Aspergillus, Synechocystis, rice, Dunaliella, and Mesembryanthemum that have been produced. The objectives of the supplemental proposal are:

* To train K-9 teachers in deciphering the function of the knocked out genes in Arabidopsis and Mesembryanthemum, particularly those genes relevant to stress tolerance and/or stress signaling. Expression will be examined under a 'gauntlet' of standardized conditions using Arabidopsis and Mesembryanthemum microarrays.

* To train K-9 teachers in identifying knockout mutations in a large number of these genes and in examining the mutant plants for altered phenotypes under the same gauntlet of conditions used for the microarray expression analysis.

To train K-9 teachers in examining the mutants for gene expression abnormalities using the Arabidopsis and Mesembryanthemum microarrays.

These results will not only provide new knowledge for future plant breeding but also new approaches for the integration of education and research using teacher training as one model. One additional benefit is that this model could be used to introduce plant science into K-9 curriculum using Arabidopsis and Mesembryanthemum as model plants. Teachers will be trained in hands-on physiological research on important and relevant biological problems using leading edge standardized technologies. The training will integrate presentation of a formal plant growth and development seminar course focusing on the technologies, participation in weekly laboratory meetings, and attendance at genomics and bioinformatics workshops and project membership meetings of this consortium such that the K-9 teachers can acquire the habits of mind gained by graduate students from the communal excitement in leading-edge scientific research that is hands-on and sustained. The central hypothesis is that teachers trained in such an environment over the long term are more likely to become life-long learners of science and therefore better able to communicate the excitement of science to their students in their classrooms. Dr. Bohnert is in discussions with Dr. Mary Williams, Assoc. Prof., at Harvey-Mudd College, CA, to establish a similar program. Dr. Hasegawa is likewise in discussions with Humboldt College, CA, and Drs. Bressan and Hasegawa have also submitted a supplemental grant (through UA) for a ROA with a High School teacher (Donola Battles) of Pittsburg, Oklahoma; this also involves screening of mutants for altered stress tolerance within the classroom.

OSU has several excellent outreach programs for K-12 teachers and minority students through the Oklahoma Partners for Biological Sciences (OPBS; Dr. Joanna Ledford, Coordinator) program funded by the Howard Hughes Foundation, the Native Americans in Biological Sciences (NABS, Dr. Kim Burnham, Director) program, and the Oklahoma Alliance for Minority Participation in Science, Mathematics, Engineering and Technology (OKAMP-SMET, Dr. Judy Batson, Manager) both funded by NSF. In collaboration with Dr. Burnham, Dr. Cushman recently hosted seven Oklahoma high-school science teachers to highlight the research capabilities and programs in the area of genomics and to encourage future interactions. The OPBS program enhances training and research opportunities for undergraduates transferring to OSU from regional two-year colleges or from high school. The NABS and OKAMP-SMET programs specifically target minority and native American student training, recruitment and retention in scientific disciplines. One native american student currently works in Dr. Pradeís laboratory. Now that formal training workshops have been developed (see below), our future goals will be to specifically target K-12 teachers and minority student participation in these training opportunities with the help of the OPBS, NABS, and OKAMP-SEMT. The teaching modules would be customized for use by high school and college levels by teachers that already participate in the OPBS, NABS, OKAMP-SMET outreach programs.

6. Specific courses, workshops and seminars Courses and workshops that are in place or planned include:

(A). Oklahoma State University John Cushman has initiated training for faculty, technicians, postdocs and students in the following genomics-related workshops and lectures: (a) 'Genomics and Bioinformatics' July 9-10, 1998; Department of Biochemistry & Molecular Biology Summer Minisymposium, Upper division course #6820, Oklahoma State University. This activity provided exposure to state-of-the-art genomics and bioinformatics research projects for over 150 faculty, postdocs, graduate students, undergraduates, and technical personnel within the state of Oklahoma and several neighboring states. (b) 'Genomics and Bioinformatics' January 6-8, 1999; Department of Biochemistry & Molecular Biology Recombinant DNA/Protein Resource Facility Workshop, Upper division course #6820. This activity provided 'hands-on' practical training in genomics and bioinformatics to 20 participants including 11 faculty members, 4 postdoctoral researchers, and 5 graduate students. Participants gained experience in high throughput, automated DNA sequencing, bioinformatics and microarray technology. (c) 'Genomics and Bioinformatics' Spring Semester 1999; Department of Biochemistry & Molecular Biology, Upper division course #6820. Nine graduate students received instruction in genome mapping and sequencing, comparative genomics, transcriptome analysis, DNA Chip technology, proteomics, functional genomics, and bioinformatics. They also gained practical training in high throughput, automated DNA sequencing, bioinformatics and microarray technology in the associated laboratory exercises. In years two and three, Cushman, Prade, and Misawa will expand the single semester course on Genomics and Bioinformatics being offered now into a two semester sequence consisting of one semester devoted exclusively to genomics and the other being devoted to bioinformatics.

(B) University of Arizona (a) Optimizing Microarray Hybridization Conditions'. June 24, 1999. This one-day workshop will feature Dr. David Ingham (Sigma Chemical Company) and focuses on printing substrates and hybridization conditions, in order to establish optimal conditions for microarray signal detection. Approximately 30 persons are enrolled for the lecture and laboratory sessions. (b) Seminars: During the spring semester several speakers at the UA plant biology program and in departmental seminar programs featured talks on various aspects of genomics. Bioinformatics was covered by Dr. Tandy Warnow (UA Dept. Computer Science) and Dr. Xiaoyin Lin (TIGR) (both on our advisory committee). Dr. Lin spent two days in discussions with various group members.

Education and Outreach: (See Training and Development Section above).

Journal Publications:

J Yale, HJ Bohnert, "Analysis of the Saccharomycescerevisiae Salt Stress Response.", PNAS, to be submitted, vol. , (), p. . to be submitted

Bohnert H, Burnap R, Bressan R, Cushman J, Galbraith D, Hasegawa PM, Misawa E, Pardo J, Prade R, Zhu J-K,, "Functional Genomics of Plant Stress Tolerance.", Plant Cell, to be submitted, vol. , (), p. . to be submitted

Book(s) or other one-time publication(s)

Cushman JC, "Genomics and Bioinformatics" , bibl. 2nd Ed. pp. 170, (1999). Teaching Manual unpublished

Internet Dissemination:


http://www.biochem.arizona.edu/bohnert/page.htm
http://www.hort.purdue.edu/cfpesp/hasegawa/hasegawa.htm
http://mendel.okstate.edu:1025/html/
http://opbs.okstate.edu/~cushman/homepage.html
addresses for homepages of group members and for the entire group

Other specific products

Approximately 20,000 lines which contain T-DNA insertions are available (May 1999), most of which are being propagated as single plants at the present time;[as of mid June, 32,120 tagged plants have been found; they are grown individually for seed propagation] T-DNA insertion lines will be disseminated through the Arabidopsis Biological Resource Center (Ohio State University).

Teaching aids

Genomics & Bioinformatics course manual; lecture notes upon request

Physical collection (samples, etc.)

cDNA libraries (multiple); EST clones; cDNA microarrays (Plant cytochrome P450 genes, 1536 M. crystallinum genes; 600 selected M. crystallinum transcripts; 120 corn transcripts with emphasis on one-carbon-metabolism). All are available upon request; fact is posted on homepages

Contributions

Contributions within Discipline:

Technology has progressed so much that the analysis of all genes in an organism becomes possible. Many projects target this objective. Our discipline, plant molecular biology, has been invigorated by a genomics approach to understanding plant function. Our contribution within the discipline is that we have begun to find and describe all genes in plants that are essential or supportive to environmental stress tolerance. We accomplish this by molecular (transcript and gene isolation), genetic (mutant generation and analysis), and functional (mutant complementation, cell biological and transgenic) studies.

After just a few months in full gear, we have identified several 100 genes whose expression is stress-dependent. As far as we know functions, there are several complexes in which genes expression responds to the stress stimulus: radical oxygen scavenging, chaperones synthesis, energy provision, and altered photosynthesis and development are foremost.

(1) Equally significant for the discipline is another finding. We have isolated a large number of transcripts, which are novel transcripts. This label is affixed to transcripts for which we are certain that they contain long reading frames (i.e., likely are not simply 3'ends of transcripts) and for which there is no homologue in either plant, fungal, bacterial or animal databases.

(2) In addition, our approach has been vindicated by the detection of a number of transcripts which appear only in transcript collections from the stressed plants (and are then abundant messages). Some of these have occasionally been sequenced also from Arabidopsis or other plant species.

(3) We have detected a number of protein coding regions which had been labeled 'hypothetical' or 'putative' in the collections of other plant species. With high homology to M. crystallinum sequences, these transcripts are not longer hypothetical.

(4) Finally, we have detected a number of transcripts that have been labeled hypothetical or putative in the genome sequence of C. elegans, D. melanogaster, and in archaebacterial genomes and human sequence databases. These have not previously been detected in other plant species.

Contributions to Other Disciplines:

If we define our discipline as salt stress responses of plants, we can relate our findings to those from other stress-related plant activities, to developmental biology and - above all - to evolutionary biology. Many stress responses have been around for a long time - they are found in fungi as well as in plants and animal systems.

An excellent justification of the project concept comes from the detection of sequences in salt-stressed M. crystallinum which are not yet found in other plant species, but have been detected in C. elegans, H. sapiens, D. melaongaster and even in some archaebacteria. In this respect our project can provide data to other disciplines.

Contributions to Education and Human Resources:

The list of collaborators indicates how many people we reach with our project in terms of training and education. We point in particular to the large number of undergraduate students working in our laboratories, including students from underrepresented groups. Further contributions have been outlined in the Project training/Development and outreach activity pages.

Contributions to Resources for Science and Technology:

At all three institutions the project has brought changes: The following characterizes the situation at all three institutions, but includes also specific aspects:

> several other groups - outside the plant area - are utilizing our microarrayer facilities;

> the biotechnology support unit (UA) has upgraded its sequencing facilities; the prize for sequencing reactions has been reduced (not enough yet, but $8 is better than $12);

> in the Dept. Computer Sciences (UA) much more emphasis is being placed on bioinformatics - with direct encouragement by the Dean of Sciences;

> administrative interest has been obtained to advance genome research effort at all three universities;

> the College of Agriculture (UA) is considering establishment of a research unit that recognizes the importance of plant genome analyses. Likewise, Purdue is actively searching for new faculty in this area;

> a much larger number of undergraduate students than before this award was made are working in plant laboratories;

> several of our colleagues have been motivated to join genomics-type activities. At UA, faculty is now involved in four of the NSF-funded genomics awards;

> there is generally more cross-departmental activity and more enthusiasm for plant research within the Biochemistry departments at UA and OSU.

> more emphasis on genomics/ genomes at all institutions;

> interest by the administration;