Rodents, especially mice, may rule supreme in the biomedical world as the model system of choice; however, there are situations in which mice may not be the ideal model system whereas an agricultural species may be. A workshop titled "Advantages of Agriculturally Important Domestic Animal Species as Biomedical Models" focused on the use of agricultural species as biomedical models. Over 100 participants from universities and federal agencies attended the workshop, held October 29-31, 2004, at Michigan State University.

A number of investigators presented their experiences with research projects that utilized agricultural species, such as chickens, pigs, cattle, and sheep, as biomedical models. Speakers were asked to discuss the following points in their presentations: the research focus and problem to be addressed; unique advantages of the domestic animal species as biomedical models; significance of findings for both human and animal health; and key elements and obstacles for successful funding.

The keynote address, "Are the domestic farm species redundant as models in biomedical research? Does mighty mouse rule supreme?", was presented by M. Roberts (University of Missouri). Other presentations discussed sheep as a model for nutritional biochemistry and prenatal programming of reproductive and metabolic dysfunction (G. Wu, Texas A&M; V. Padmanabhan, University of Michigan). Cattle were presented as a model for stem cell biology, immunobiology, and pathogen transmission (H. Lewin, University of Illinois; M. Jutila, Montana State University; G. Palmer, Washington State University; V. Kapur, University of Minnesota). The chicken, although not a mammal, also serves as a model for investigations in reproductive biology and diseases (J. Bahr, University of Illinois). Pigs are used for biomedical research on the cardiovascular system, cardiac arrhythmias, nutrition research, xenotransplantation, and adipose development and metabolism (R. Prather, University of Missouri; H. Laughlin, University of Missouri; R. Gilmour, Cornell University; J. Odle, North Carolina State University; D. Hausman, University of Georgia). The pig represented the most common model species, due to the similarity in anatomy and physiology between pig and human organs.

This workshop did not present a comprehensive representation of the roles that agricultural species play in biomedical research, but merely a sampling of successful research projects. Some common themes that emerged from the presentations included the perception that reviewers are not only unaware of alternative models, but may be biased against agricultural species. Strategies to counteract reviewer bias focused on a number of points, such as educating grant reviewers on the merits of agricultural species as a more appropriate model system. Furthermore, reviewers should be made aware that agricultural models have unique aspects that can be utilized to pioneer research to address gaps in knowledge of human health, not just confirm that of rodent models—i.e., the agricultural species may be a more suitable model than rodents, not just an alternative model.

Other obstacles discussed included the need for a critical mass of investigators, the lack of availability of reagents and tools, and the costs of doing research. Compared to mice, agricultural species lack comparable database resources, genomic information, and tools. For example, NIH funds $100M/year for pig research, yet the pig genome has yet to be sequenced. Chicken and bovine genomes have been sequenced, although neither is at the finished stage comparable to the human or mouse genome. For chickens, lack of an efficient method for generating transgenic chickens is a major obstacle to research. The high cost for purchasing, housing, and feeding agricultural species is an additional hindrance for all research with agricultural species. In addition, animal variability often necessitates use of greater numbers of animals to obtain statistically significant results, further driving up total cost. However, on the positive side, the large quantity of tissue available from agricultural species offers an advantage over rodents.

Grantsmanship for research on agricultural species was another topic of the workshop. Grant writing success generally follows the same formula, regardless of funding source. Good science is valued, first and foremost, and solid preliminary data are essential to success in grant funding. In some cases, investigators are able to use smaller USDA grants to obtain preliminary data that allowed them to compete for larger NIH grants. Senior investigators need to have a strong track record of publication in biomedical journals. Publishing in only the Journal of Animal Science or Poultry Science does not always reach the intended biomedical community.

Small groups of workshop participants addressed additional questions in two breakout sessions. Breakout Session I discussed the following questions:

Discussants in Breakout Session I determined that perceived barriers to using agricultural species as biomedical models were similar to those outlined by the presenters, such as availability of tools and reagents, knowledge and appreciation of agricultural models, adequacy of facilities, animal costs, institutional support, and ethical issues. Further, they suggested that the unique knowledge to be generated with agricultural species will help in understanding complex traits, population dynamics, epidemiology, and comparative functional genomics. There was the perception that use of domestic animals requires more justification because of the lack of appreciation of the models and understanding of the barriers. Therefore, more preliminary data may need to be submitted to convince reviewers of the merits of agricultural models.

In Breakout Session II, further questions were discussed:

• What is needed to enhance the use of domestic species as biomedical models?

Some suggestions for enhancing use of domestic species as biomedical models included the following: increasing awareness of benefits of agricultural models, perhaps through symposia at biomedical meetings; increasing resources for databases and animal facilities; and developing grants for training integrative biologists. Another proposed strategy is the establishment of databases for central sharing of resources—the availability of mouse lines and cells and molecular biology reagents has greatly facilitated research with mice and would do the same for the agricultural species. The development of a joint NIH and USDA Request for Application (RFA) for alternative biomedical models would also enhance the use of agricultural species. Cooperation between medical schools and animal science departments could be strengthened by joint medical and agriculture school appointments and appropriation of institutional funds for interdisciplinary research. In addition, the physical (different campuses) and programmatic (different goals and research cultures) disconnect between agriculture and medical school programs needs to be addressed.

Introduction
Eukaryotic genes are regulated by a hierarchy of intricate controls that include modifications to chromatin structure. Within the primary chromatin fiber, DNA is packaged into nucleosomes by core histones. Posttranscriptional modification of nucleosomal histones, particularly through acetylation and deacetylation, is an important mechanism in the regulation of eukaryotic gene expression. Acetylation of the histones by acetyltransferases leads to a relaxed association, and thus, enhances accessibility of promoters to components of the transcription machinery and activates gene expression. Deacetylation of histones by histone deacetylases (HDACs) removes acetyl groups from the core histones. A reduction in the level of histone acetylation condenses chromatin, which obstructs transcription machinery access to gene regulatory elements, resulting in repression of transcription.

Different classes of HDACs have been identified in eukaryotes, including yeast, animals, and plants. Class I and II HDACs are homologous to yeast RPD3 and HDA1, respectively. In plants, functional studies indicate that RPD3 may provide housekeeping HDAC activity. Class III HDACs are related to the yeast silencing protein SIR2 and are dependent on nicotinamide adenine dinucleotide (NAD) for enzymatic activity. In addition to these three classes of HDACs, plants possess another type of HDAC, namely, the HD2 family. The HD2 family appears to be unique and unrelated to other classes of HDACs and shares some sequence similarity with the FKBP-type PPIases. Four members of HD2 are identified, HD2A, 2B, 2C, and 2D.

The role of histone deacetylases in transcription repression has been described in yeast and animals. In plants, involvement of histone deacetylases in the control of gene expression has not been studied. Here we report transcriptional repression by HDACs in plants and describe a system of using HDACs for repression of transgene expression. We explored proteins of HD2 and RPD3 classes of HDACs for repression and used yeast transcription factor, GAL4, or a plant transcription factor, to target an HDAC protein to the gene to be repressed. The study demonstrates that HDACs can effectively repress the expression of a transgene in plants.

Repression of expression via transient gene expression
Repression of gene expression by HDAC proteins was first tested via transient gene expression. A reporter vector was constructed in which a GUS reporter gene was driven by a strong constitutive promoter. Additionally, two copies of GAL4-binding sites (UASGAL4) were placed in front of the promoter in the reporter vector (Fig. 1). A second vector, effector, was also constructed. The effector contains an HD2 protein, HD2A, from Arabidopsis, and the protein was fused in frame with the DNA-binding domain of the yeast transcription factor GAL4 (GAL4BD). The fused protein was driven by another constitutive promoter (Fig. 1). It is expected that the reporter gene is repressed by GAL4-HD2A protein, which would target a promoter containing the GAL4-binding sites (UASGAL4). The reporter and effector plasmids were co-introduced into tobacco leaves by microprojectile bombardment. Analysis of transient GUS expression showed that HD2A proteins strongly repressed GUS gene expression when compared with expression of the reporter plasmid alone or in combination with a control effector plasmid that expressed the GAL4 protein only (Fig. 1). Different types of HDAC proteins, including HD2A, 2B, 2C, and RPD 3A were tested in the same manner and all these HDAC proteins exhibited repression. This suggests that HDACs can mediate transcriptional repression of a targeted reporter gene in vivo.

Figure 1. (A) Schematic diagram of the reporter and effector constructs. The reporter construct contains an activation sequence (binding site), such as yeast UASGAL4 or plant GCC box, which are placed in front of a promoter driving the GUS gene. The effector construct contains DNA binding domains (DNA-BD), such as the yeast GAL4 DNA binding domain (GAL4BD) or the plant transcription factor Pti4 gene binding domain. The DNA binding domain is fused to a HDAC protein. T: terminator. (B) Repression of GUS gene expression by Arabidopsis HD2 genes (AtAHD2A, AtHD2B, and AtHD2C). The reporter vector containing UASGAL4–Promoter-GUS was co-bombarded with an effector vector containing Promoter-GAL4BD-HD2A, HD2B, or HD2C respectively. [Used with permission; Blackwell Publishing1]

Repression of gene expression in transgenic plants
The expression repression by HDACs was further evaluated in transgenic plants. UASGAL4-Promoter-GUS reporter construct (Fig.1) was used to transform Arabidopsis to generate reporter lines, which were selected by kanamycin. Effector lines were generated by using the effector plasmid, Promoter-GAL4/HD2A (Fig. 1) and generated by hygromycin selection. The reporter lines showing high levels of GUS activity were crossed with effector lines that showed strong GAL4/HD2A expression. F1 seedlings that were resistant to both kanamycin and hygromycin were selected for further evaluation. Analysis showed that GUS gene expression in F1 progeny plants containing both effector and reporter constructs was significantly reduced compared with plants that contained the reporter DNA only. Northern analysis indicated that only a small amount of GUS transcript was detected in F1 progeny. Decreased GUS transcript in F1 progeny was correlated with strong GAL4/HD2A expression in these plants. This demonstrates that a gene repression system using HDAC protein is applicable in transgenic plants with stable gene expression.

Repression of gene expression in a specific manner
Directing the repression of gene expression to a particular tissue or at a particular time is desirable and will have wide application. To test if repression can be achieved in a specific manner, a tissue-specific promoter was explored in the system. Napin promoter (NAP2), the seed-specific promoter, replaced the constitutive promoter to drive the GAL4/HD2A fusion gene. Transgenic Arabidopsis plants containing effector or reporter constructs were generated separately. Effector plants and reporter plants were crossed and F1 seedlings that were resistant to both kanamycin and hygromycin were selected. Analysis showed that GUS expression was repressed only in seeds, not in other organs, of the F1 hybrid progeny. Results indicate that specific repression by HDACs can be achieved by directing expression of GAL4/HD2 protein in a particular manner as desired.

Gene repression through interacting with a plant transcription factor
These results demonstrate that an HDAC protein can repress gene expression, and the repression is directed by yeast GAL4 binding system. We were also interested to know if HDACs can mediate gene repression through interacting with a plant transcription factor. We tested a plant Pti4/GCC box interaction system for repressing target gene expression. Pti4 is a tomato transcription factor that belongs to the ERF (ethylene-responsive element binding factor) family of proteins, which can regulate the expression of GCC box-containing genes. The effector construct was made that contained an HDAC gene (HD2A) fused with the Pti4 protein DNA binding domain (Pti4BD). The reporter vector was constructed in which five copies of GCC box were placed in front of the promoter that drives the GUS gene (Fig.1). The reporter vector and the effector vector were co-introduced into tobacco leaves by bombardment, anticipating that the Pti4 gene will direct an HDAC protein to a promoter containing a GCC box. Results showed that the expression of the GUS reporter gene containing the GCC box was significantly inhibited when co-introduced with the Pti4/HD2A fusion gene. The results demonstrate that HDACs can mediate gene repression through interacting with a plant transcription factor. Expression repression using plant transcription factor binding domain can lead to possibilities of mediating expression of genes residing in the genome without cloning and alteration of their sequence.

Summary
Chromosome remodeling is an important factor in regulating eukaryotic gene expression. Histone modification via acetylation and deacetylation is particularly important in chromosome configuration. We demonstrated gene repression in plants by HDACs and described a technology for repression of gene expression by HDAC proteins. Repression or altering of gene expression via chromosome remodeling using HDACs provides a useful and alternative avenue for gene silencing. The system is useful for assessment of gene function, for manipulation of biochemical processes, and for control of plant developmental processes.

1. Zhou C, Labbe H, Sridha S, Wang L, Tian L-N, Latoszek-Green M, Zhang L, Brown D, Miki B, & Wu K (2004) Expression and function of HD2-type histone deacetylases in Arabidopsis development. Plant J. 38, 715-724

2. Wu K, Tian L-N, Brown D, & Miki B (2003) Repression of gene expression in transgenic plants using a HD2 histone deacetylase. Plant J. 34, 241-247

3. Lusser A, Brosch G, Loidl A, Haas H, & Loidl P (1997) Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein. Science 277, 88-91

4. Murfett J, Wang X, Hagen G, & Guilfoyle TL (2001). Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression. Plant Cell 13, 1047-1061

TILLING: HARVESTING FUNCTIONAL GENOMICS FOR CROP IMPROVEMENT
Charles Paul (Max) Moehs

Since the first publication describing the technique of TILLING (Targeting Induced Local Lesions in Genomes) in 2000¹, this method of reverse genetics has been widely adopted by the academic community for use in model organisms, including Drosophila, zebrafish, and Arabidopsis. Now a recent paper by Slade et al.² highlights its potential for crop improvement.

Slade et al. describe the application of TILLING to the identification of an allelic series of variants in the granule-bound starch synthase I (GBSSI) gene in hexaploid and tetraploid wheat. GBSSI or Waxy plays a critical role in the synthesis of amylose, which, in addition to amylopectin, comprises the starch fraction of the seed. Reduction or loss of GBSSI function results in starch with a decreased or absent amylose fraction, which is desired for its improved freeze-thaw stability and resistance to staling compared to conventional starch.

Prior to Slade et al.’s work, wheat breeders had developed breeding lines of waxy wheat, although no waxy wheat is yet grown commercially. The conventional breeding approach, however, took considerable effort over many years to find rare spontaneous knock-out alleles of GBSSI in germplasm collections of wheat landraces using time-consuming phenotypic screens such as SDS-polyacrylamide gel electrophoresis of waxy proteins. The search for natural knock-out alleles of GBSSI in bread wheat is complicated by the presence of A, B, and D genomes in allohexaploid bread wheat and A and B genomes in tetraploid pasta wheat. GBSSI is encoded by a locus found on the 7A and 7D homoeologous chromosomes, and, due to a translocation, on the 4B chromosome. In addition to the difficulty of finding naturally-occurring knock-out alleles of the waxy locus, plant breeders have had to contend with the necessity of removing associated unadapted traits from landraces when introgressing the recessive waxy trait into elite wheat germplasm.

In their paper published in Nature Biotechnology, Ann Slade and her colleagues at Anawah, a company focused on the commercial applications of TILLING, use the identification of extensive allelic series of the waxy genes in both hexaploid bread wheat and tetraploid pasta wheat as a demonstration of the power of TILLING for practical crop improvement. TILLING is an updated version of mutation breeding, a technique that has been practiced for decades by plant breeders. TILLING differs from mutation breeding, however, in the method of mutation detection: while traditional mutation breeding has been used primarily for readily observable phenotypes, such as plant height or disease resistance, TILLING detects the mutation directly in the DNA sequence of the gene of interest. Because the mutation is not required to have an immediately observable phenotype, TILLING is a method of reverse genetics that can be used to test hypotheses about the functions of any desired candidate gene.

The basic procedure of TILLING is straightforward; it involves 1) the creation of a large genetically diverse population of plants; 2) the high throughput identification of individual plants whose genotype predicts a phenotype of interest; and 3) the evaluation of these individuals’ phenotypes for the accelerated development of novel cultivars that exhibit traits of interest. It combines random mutagenesis of seeds with an alkylating agent such as ethyl methanesulfonate (EMS) or other mutagenic agent with the targeted identification of induced alterations in the genes of interest.

Chemical mutagenesis with alkylating agents produces a high density of point mutations, including missense and nonsense mutations. The combination of the high density of mutations in an individual plant with a large population of plants results in the creation of multiple lesions in any desired gene. These lesions are subsequently identified by screening the DNA of the M2 progeny of the mutagenized seeds. PCR is used to amplify genes of interest with fluorescently labeled gene-specific primers in pools of DNA from M2 plants, and then mismatch-containing duplexes are created by melting and reannealing the PCR fragments. The heteroduplex DNA is identified by cleaving the mismatch with a specific mismatch-recognizing endonuclease³ followed by sequence analysis. Finally, phenotypic analysis of the selected plant and its progeny is carried out. The selected plant serves as the parent in subsequent breeding generations, and the segregation of the desired SNP can be followed with molecular markers⁴.

A key parameter in any TILLING project is the mutation frequency achieved in the TILLING population. In this regard, the work by Slade and colleagues is unique since they found a mutation frequency of one in 24 kb in hexaploid wheat and a frequency of one in 40 kb in tetraploid wheat. This is approximately five fold higher than had been observed in Arabidopsis. Slade et al. report that their mutagenized population showed few apparent visible phenotypes attributable to the mutagenesis, and they hypothesize that this is due to genetic redundancy inherent in this polyploid crop. Because of this remarkable mutation frequency, Slade et al. identified 196 new alleles in the A and D genome waxy genes in only 1,152 individual plants screened in their hexaploid TILLING population, and 50 new alleles in only 768 individuals in their tetraploid pasta wheat TILLING population. These allelic series in both hexaploid and tetraploid wheat included multiple truncation and splice junction mutations as well as numerous missense mutations with predicted deleterious effects on the function of the waxy enzyme.

One plant was found to have a truncation mutation in the D genome locus as well as a missense mutation predicted to severely affect enzyme function in the A genome homoeolog. Because the Express cultivar used to generate the TILLING population has a naturally occurring deletion of the B genome homoeolog, progeny of the plant having induced mutations in the D and A genome homoeologs included homozygous triple mutation-containing plants. Iodine staining of one of these plants confirmed the near-null expected waxy phenotype.

These new alleles in GBSSI in wheat represent a useful resource for breeding a range of waxy and partial waxy wheats, but even more importantly, this work represents proof-of-concept for TILLING other genes whose modification may be desired in wheat or other crops. In order to identify new alleles via TILLING in wheat, homoeolog-specific primers are required. In the case of Waxy, the sequences of the three homoeologous sequences were already known, which facilitated primer design. Future wheat TILLING of other genes is likely to require additional molecular research to develop homoeolog-specific target primers and will be more complex for genes that are members of gene families.

Ann Slade and her colleagues at Anawah have convincingly shown that TILLING is a technique with considerable potential for crop improvement. It represents an extension of the use of spontaneous and induced mutants in plant breeding and allows the direct identification of beneficial nucleotide and amino acid changes in genes with known functions and their use as the genetic markers for selection. The range of alleles that can be developed via TILLING in a short time is unparalleled and unlikely to be found elsewhere in the pool of germplasm accessible to plant breeders (including landraces and undomesticated relatives). Because the TILLING population is a permanent resource, the results of basic scientific research can be efficiently translated into crop improvement as new information about the functions of potential gene targets becomes available.

1. McCallum CM et al. (2000) Targeting induced local lesions in genomes (TILLING) for plant functional genomics. Plant Physiology 123, 439-442

2. Slade AJ et al. (2005) A reverse genetic, non-transgenic approach to wheat crop improvement by TILLING. Nature Biotechnology 23, 75-81

3. Oleykowski CA et al. (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Research 26, 4597-4602

4. Neff MM et al. (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. The Plant Journal 14, 387-392

TOBACCO AND RICE PLANTS EXPRESS INSULIN-LIKE GROWTH FACTOR
P. Janaki Krishna

The insulin gene family, comprised of insulin, relaxin, and insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), represents a group of structurally related polypeptides whose biological functions have diverged. Of these, human insulin-like growth factor 1 (hIGF-1) is essential for cell growth and intrauterine development. hIGF-1 is used therapeutically in treating growth hormone sensitive syndrome (Laron syndrome), type A insulin receptive syndrome, diabetes, osteoporosis, and acquired immune deficiency syndrome (AIDS). The recombinant human insulin-like growth factor 1 (rthIGF-1) is likely to be effective in slowing disease progression in people with amyotrophic lateral sclerosis, known in some countries as "Lou Gehrig’s disease."

Several efforts have been made to express rthIGF-1 in various host-vector systems, including yeast and transgenic rabbits. However, these studies report some disadvantages in working with animal systems, such as low transgene expression levels and contamination of animal-based products with human pathogens. To circumvent these difficulties, plant-based vector systems for production of recombinant proteins have been tested by many researchers, as they present a minimal risk of contamination by pathogenic microorganisms. The most suitable targeted edible tissues for expressing therapeutic plant proteins are cassava and potato tubers, corn and rice grains, and banana and tomato fruits. The successful expression of human recombinant proteins in different transgenic plants such as barley, banana, tobacco, maize, and carrot has also been demonstrated.

Researchers from the Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Canada, and the Steacie Institute for Molecular Sciences, National Research Council of Canada, have expressed rhIGF-1 in rice. The team used two coding sequences—one with the original human sequence, but partially optimized for expression in E. coli, and the other with a plant codon optimized sequence for obtaining higher levels of expression in plant systems. Three hIGF-1 recombinant expression cassettes were constructed using maize ubiquitin promoter. The production plasmid pBKIGF-2B containing the LAM B-IGF-1 coding sequence was partially optimized for expression in E. coli. In addition, efforts were made to augment expression levels in rice by incorporating a rice prolamin signal sequence in one construct to target the protein to the endoplasmic reticulum. The new construct, designated pUSYN-IGF-1, was designed for transformation purposes and contained the maize ubiquitin-1 promoter, rice prolamin signal sequence, plant codon optimized hIGF-1 coding sequence, and Nos-TER sequence. To obtain higher levels of rthIGF-1 expression in plants, the plant codon optimized hIGF-1 coding sequence along with 66 bp that encoded the rice 13kDa prolamin signal sequence was also ligated into pAHC25. The engineered plasmids were then used to transform Agrobacterium strain LBA4404. In tobacco, the Agrobacterium-mediated method was used to transform Nicotiana tabaccum cultivar Xanthi. In rice, the pKHG4 plasmids containing the recombinant expression constructs were used to transform Japonica rice (Oryza sativa cultivar 93VA) through the Agrobacterium-mediated method. Later these plants were transferred to a greenhouse until maturity.

Approximately 20 independent transgenic plants were generated for each transgenic line and screened by ELISA. The data confirmed that all transformed plants produced rthIGF-1 up to a level of 241 ng/mg of total protein in tobacco and 371 ng/mg of total protein in rice. The biological assays conducted using protein extracts from all the transgenic plants showed that rthIGF-1 was effective in stimulating in vivo growth and proliferation of human SH-SY5Y neuroblastoma cells, indicating that plant-produced rthIGF-1 was stable and biologically active. However, ELISA data indicated that, although both rice and tobacco plants were transformed with identical expression constructs, higher levels of rthIGF-1 in transgenic rice were obtained compared to transgenic tobacco. Transgenic plants showing the highest expression levels were selected for further studies.

Thus, this study demonstrates that engineering expression cassettes with strong plant promoters, signal sequences, and proper codon choice is essential to produce large amounts of recombinant human protein in plants. The investigating team further showed the feasibility of producing biologically active rthIGF-1 protein over one generation of transgenic tobacco plants using an E. coli-derived unmodified expression construct. By increasing the GC content of the hIGF-1 coding sequence from 52% to 70%, and with the addition of a plant signal sequence, the investigators improved recombinant protein accumulation in transgenic plants. The study also suggested that a monocot promoter in a ‘native’ monocot plant led to significantly higher yields of rthIGF-1. If the full beneficial potential of rthIGF-1can be achieved, it is expected that the future demand for production of these plants of clinical importance will increase considerably.

In conclusion, the study further establishes that transgenic plants could be developed as useful tools to express foreign proteins for further clinical applications. Also, where high amounts of recombinant human protein are in demand, these proteins can be produced in either greenhouse or field-grown crops. In other words, this study further confirmed that plants can be used as bioreactors for producing substances of economic, industrial, and clinical importance.

1. Panahi M et al. (2004) Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants. Transgenic Research 13, 245-259

2. Brem G et al. (1994) Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits. Gene 149, 351-355

3. Horvath H et al. (2000) The production of recombinant proteins in transgenic barley grains. Proc. Natl. Acad. Sci. USA 97, 1914-1919

4. Leite A et al. (2000) Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants. Mol. Breed. 6, 47-53

UNWINDING AFTER HIGH SALINITY STRESS: DEVELOPMENT OF SALINITY TOLERANT PLANT WITHOUT AFFECTING YIELD
Narendra Tuteja

Summary
Salinity has a considerable effect on world agriculture, significantly reducing productivity of agricultural plants. In response to salinity stress, a series of genes, such as those encoding for ion channels and osmolytes, are upregulated to help mitigate damage. Because DNA helicase from pea protects plants from stress induced by high salt concentrations, Dr. Narendra Tuteja and colleagues surmised that DNA helicases must also be upregulated in response to high salinity stress¹. DNA helicases are enzymes responsible for unwinding DNA, a necessary step in gene transcription, and thereby are involved in maintaining genome integrity. Using a pea plant model, the researchers found that pea DNA helicase 45 (PDH45) transcript is increased three-fold in response to high salt concentrations. The helicase was also upregulated in response to other stresses, such as dehydration and low temperatures. To examine the function of PDH45, Dr. Tuteja’s group developed a transgenic tobacco plant containing the gene. Wild type tobacco plants suffered in response to high salt, but transgenic plants overexpressing PDH45 protein continued to grow. In addition, subsequent generations of transgenic tobacco plants maintained the exogenous gene and continued to resist high salt stress, suggesting that stress tolerance can be manipulated in crop plants. Overexpression of DNA and RNA helicases provides a possible example of the exploitation of DNA/RNA unwinding pathways for engineering salinity tolerance without affecting yield in crop plants.

Introduction
Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. Due to over-irrigation, salinity, which hampers metabolic activities in plant cells, is increasing in many areas around the world. Plants respond to stress by altering the expression of many genes, leading to adaptation and survival². Because salinity stress affects cellular gene expression machinery, it is evident that molecules involved in nucleic acid processing, including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination, and transcription, while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis, and translation initiation³. Most helicases are members of the DEAD-box protein super-family⁴. The eukaryotic translation initiation factor (eIF-4A) is a prototypic member of this family.

We have previously reported the isolation of pea DNA helicase 45 (PDH45), which exhibits striking homology with eIF-4A and contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities⁵. Here we report the upregulation of PDH45 in response to abiotic stresses. Its overexpression in tobacco plants confers increased tolerance to salinity, indicating its potential for improving stress tolerance in crop plants.

Results
PDH45 is induced by salinity and other stresses in pea. In order to study the effect of salt stress on expression of the PDH45 gene, 7 to 8 day old pea seedlings were exposed to 50 – 300 mM sodium chloride (NaCl), and transcript levels were analyzed. In leaf and root tissues, a significant induction of PDH45 mRNA was observed after 12 h exposure to NaCl. Western blot analysis of pea leaves and root tissue stressed with NaCl also showed a time-dependent accumulation of PDH45 protein following up to 12 h of stress treatment. This effect was specific to Na⁺, as lithium chloride (LiCl) did not result in significant induction in transcript level. Inducibility of PDH45 transcript was also observed in dehydration, wounding, low temperature (4°C), and ABA stresses.

Development of salinity tolerant tobacco without affecting yield. Overexpression of PDH45 in tobacco plants, driven by a constitutive CAMV-35S promoter, confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. T0 transgenic plants showed high levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield in terms of seed weight. No major difference in overall performance or total seed yield of transgenic plants grown in 200 mM NaCl was found. Although transgenic seedlings exposed to continuous stress of 200 mM NaCl in soil showed a slightly slower growth rate initially (Fig. 1, upper panel) compared to similar plants grown without stress, they continued to grow, reached maturity, flowered, and set seeds. On the other hand, growth of WT seedlings was severely affected by continuous exposure to salinity stress, and they could not survive under these conditions (Fig. 1, lower panel). Significantly, pod size, seed size, seed number, and seed weight were statistically similar to those from WT plants grown without stress. Measurement of Na⁺ ions in different parts of the plant showed higher accumulation in old leaves and negligible accumulation in seeds of T1 transgenic lines as compared with WT plants. In addition, subsequent generations of the transgenic tobacco plants maintained the exogenous gene and continued to resist high salt stress, suggesting that stress tolerance can be manipulated in crop plants.

Discussion and hypothesis of salt tolerance by helicase
Implementation of biotechnology strategies to improve salinity tolerance requires that research identify salt tolerance effectors and the regulatory components that control them during the stress episode¹. Our research has provided a new insight into plant tolerance to salinity by identifying a DEAD-box helicase, PDH45, as a novel genetic determinant of salt tolerance and yield stability. The PDH45 transcript was also upregulated by other abiotic stresses, which suggests that the increase could be due to water stress resulting from salinity- and mannitol-induced desiccation. Induction of transcript was stimulated by the phytohormone, ABA, which modulates gene activation and repression under multiple environmental stress conditions, such as drought, cold, and salinity. We observed that a single copy transgenic line with low PDH45 levels (Fig. 1) also exhibits salt tolerance, clearly indicating that position of integration or copy number does not influence expression pattern under stress. This also indicates that there must be a minimum threshold of overexpressed protein above the untransformed levels that is sufficient to confer tolerance.

Figure 1. Salinity tolerance of tobacco transgenic plant (T1 generation) overexpressing pea DNA helicase 45 (PDH45)1. Top image shows the phenotype of WT and transgenic seedlings growing in soil pots supplied with water or 200 mM NaCl solution fortnightly. Bottom image shows the WT and transgenic plants in soil pot supplied with 200 mM NaCl solution. Note that WT plant could not sustain growth under salinity stress.

The exact mechanism of PDH45-mediated tolerance of salinity stress is not understood. However, based on the properties of PDH45 that were studied earlier⁴, and those known for DEAD-box proteins^2,3, we suggest two sites of action of this dual helicase: 1) it may act at the translation level to enhance or stabilize protein synthesis; or 2) it may associate with DNA multi-subunit protein complexes to alter gene expression. Regarding support for the first hypothesis, we have previously demonstrated that antibodies against PDH45 inhibited in vitro protein synthesis, which suggests its role in translation⁴. It is evident that mRNA and protein synthesis are very sensitive to stress, so factors involved in transcription and translation are potential targets of salt toxicity in plants. In bacteria, the toxic effect of Na⁺ is mainly in translation rather than in RNA synthesis. Mechanisms of translation initiation are conserved among eukaryotes, and regulation of translation occurs at the step of initiation. We envisage the PDH45 function, considering that it is homologous to eIF-4A and contains RNA helicase activity⁴. The RNA helicase activity of PDH45 could facilitate transcription by altering the structure of nascent RNA, a process that can stimulate reinitiation and/or elongation. The second hypothesis is supported by the demonstration of the interaction of PDH45 with topoisomerase I⁴. This interaction is proposed to play an important role in regulating chromatin structure during RNA polymerase II-mediated transcription. PDH45 may have an important role at the level of transcriptional regulation.

In this report, we provide direct evidence for the involvement of a DEAD-box helicase (PDH45) in conferring salinity tolerance in transgenic tobacco plants, thus suggesting a new pathway for manipulating stress tolerance in crop plants. This also provides a possible example of the exploitation of DNA/RNA unwinding pathways for engineering salinity tolerance without affecting yield in crop plants.

1. Sanan-Mishra N, Pham XH, Sopory SK, & Tuteja N. (2005) Pea DAN helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. PNAS 102(2), 509 - 514

2. Hasegawa PM, Bressan RA, Zhu J-K, & Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463-99

3. Tuteja N, & Tuteja R. (2004) Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery. Eur. J. Biochem. 271(10), 1835-48

4. Rocak S, & Linder P. (2004) DEAD-box proteins: the driving forces behind RNA metabolism. Nat. Rev. Mol. Cell Biol. 5, 232-241

5. Pham XH, Reddy MK, Ehtesham NZ, Matta B, Tuteja N. (2000) A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity. Plant J. 24, 219-229