The advent of agricultural biotechnology has raised many biosafety concerns over the past decade. One of the more interesting concerns has been the potential for horizontal gene transfer (HGT). The movement of a transgene from plant to microbe could pose a significant risk, especially if an antibiotic resistance gene, originally from a bacterium, could be transferred to a pathogenic bacterium, causing new antibiotic resistance problems for human health. These concerns have prompted regulators and companies alike to look askance at the use of antibiotic resistance genes in transgenic plants.

Researchers have expended much energy and resources into developing alternative transgenic plant production schemes such as herbicide tolerance, positive selection¹, and marker-free selection². All these alternatives have prominent drawbacks. After all, approximately 70% of all transgenic plants have been produced using the neomycin phosphotransferase II (nptII) gene from Escherichia coli for good reason: it works, especially in most dicot species.

Besides alternative selection technologies, research has also focused on numerous technologies to remove markers using site-specific recombination³. While site-specific transgene removal might well be the ultimate solution to minimizing exogenous DNA in transgenic plants, it is far from routine in today’s laboratory. Thus, most researchers and smaller companies still use antibiotic selectable markers to produce transgenic plants for at least two reasons: 1) the high efficiency of using antibiotic selection; and 2) intellectual property constraints and technology availability of other techniques. Thus, new, and perhaps safer, selectable markers using established and proven selection schemes would be attractive for many plant biotechnologists.

We recently described a plant gene that confers kanamycin resistance to transgenic plants⁴. In this article we will discuss the relevance of this new antibiotic resistance gene to discussions of HGT biosafety, public acceptance and regulatory concerns, and its comparison to nptII. Many people contend that nptII is safe, but others have recently argued that if HGT occurred one trillion-fold less often than current risk assessment literature presumes, HGT could still have negative impacts⁵. Thus, the concern over HGT, especially of antibiotic resistance genes, perhaps warrants a closer look.

There are dozens of ATP binding cassette (ABC) transporters in Arabidopsis thaliana, and the discovery of a unique function of the ABC transporter Atwbc19 was quite accidental. We performed microarray experiments in which A. thaliana was exposed to the explosive chemical, trinitrotoluene (TNT)⁶. Atwbc19 was one of several upregulated genes revealed, and we decided to perform additional experiments to assess the opportunity to use this gene in transgenic plants for potential explosives detection and remediation⁶. We noticed that a T-DNA insertional knockout mutant did not grow in media containing kanamycin, so we decided to produce transgenic plants with the ABC transporter, with and without an nptII cassette, to test whether Atwbc19 could be used as an nptII substitute. We found that its conferred resistance to kanamycin (and only kanamycin) was similar to nptII in transgenic plants, thus leading to the characterization of the first plant gene endowing resistance to antibiotics⁴. The initial experiments were in tobacco, but we are far enough along in experiments to produce transgenic Brassica species to conclude that it is effective in this genus as well.

The availability of the Atwbc19 gene could enable efficient and safe (with regards to HGT) production of transgenic plants. After all, this gene has presumably been in plants for eons, and there is no evidence that it has been transferred to bacteria during the course of evolution. Bacteria, like all organisms, have ABC transporters, but database searches have yielded no ABC transporter gene hits with plant-like codon use patterns in bacteria. Indeed, this illustrates one conceptual hurdle in the entire plant-to-microbe HGT argument: there simply are not many examples of plant-like genes found in bacteria, indicating that HGT from plants to microbes is quite rare. The inverse is true, however; that is, there are well documented examples of bacteria-like genes (and indeed entire microbial symbionts) being co-opted into the plant nuclear, chloroplast, or mitochondrial genome. And this is no surprise really—after all, Agrobacterium tumefaciens and its relatives naturally transform plants inserting bacterial DNA⁷.

So, this begs the question, how serious is the threat of HGT from transgenic plants to bacteria? Transgenic plant-to-microbe HGT has been shown to occur under experimental conditions when the bacteria already contained a form of the plant transgene (experimental tricks—see reference 7 for a discussion), but gene transfer from transgenic plants to bacteria has never been shown occurring in the field⁷. Nonetheless, there is a lack of data on the real rates of HGT between transgenic plants and microbes, and hence prestigious groups around the world, such as WHO, FAO, and the NAS, are urging researchers to produce transgenic plants without antibiotic resistance markers⁸. Does the availability of the plant Atwbc19 change anything, or should it be grouped, biosafety risk-wise, with nptII and other selectable markers of bacterial origin?

While the absolute risk of HGT is unknown, we believe there are at least four reasons why Atwbc19 would carry relatively less risk than nptII and other antibiotic resistance genes of bacterial origin. First, Atwbc19 is very specific for kanamycin. Unlike nptII, it does not confer resistance to geneticin, neomycin, or other aminoglycoside antibiotics⁴, which are used clinically more often than kanamycin. Second, at 2.2 kb, Atwbc19 is approximately 2.75 times larger than nptII; thus the chances of it being integrated intact into a bacterial recipient would, at least, be that much lower. Third, unlike genes of bacterial origin, Atwbc19 has plant codon usage. Thus, if it were to be introgressed into a bacterial genome, it would likely be expressed less than a bacterial gene. Finally, and perhaps most importantly, even if Atwbc19 were transferred into bacteria and were expressed, it might not lead to an antibiotic resistant phenotype. This particular factor is not mentioned in the HGT literature. Our hypothesis is that Atwbc19 is targeted to the vacuole membrane of the plant cell, and its mode-of-action involves the active transport of kanamycin into the vacuole where it is sequestered⁴. While our data are equivocal with regards to precise targeting, this mode-of-action is consistent with published data on other ABC transporters. If this were, indeed, the mode-of-action, we would expect that Atwbc19 would not confer kanamycin resistance to bacteria since they lack a prominent central vacuole for sequestration of toxins. The same is true for mammalian cells. We have unpublished preliminary data showing that kanamycin resistance is not conferred to Escherichia coli when the gene is placed under the gal promoter, and we are following-up these findings with formal experiments with this and other bacterial species and mammalian cell cultures.

1. Reed J et al. (2001) Phosphomannose isomerase: an efficient selectable marker for plant transformation. In Vitro Cell Dev Biol-Plant 37, 127-132

2. de Vetten N et al. (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21, 439-442

3. Baszczynnski CL et al. (2003) Site-specific recombination systems and their uses for targeted gene manipulation in plant systems, pp157-178 in Stewart CN Jr (ed) Transgenic Plants: Current Innovations and Future Trends. Horizon Scientific Press: Wymondham, UK

4. Mentewab A & Stewart C N Jr. (2005) Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat Biotechnol 23, 1177-1180

5. Heinemann JA & Traavik T (2004) Problems in monitoring horizontal gene transfer in field trials of transgenic plants. Nat Biotechnol, 22, 1105-1109

6. Mentewab A, Cardoza V & and Stewart CN Jr. (2005) Genomic analysis of the response of Arabidopsis thaliana to trinitrotoluene as revealed by cDNA microarrays. Plant Sci 168, 1409-1424

7. Broothaerts W et al. (2005) Gene transfer to plants by diverse species of bacteria. Nature 433, 629-633

8. Davison J (2004) Monitoring horizontal gene transfer. Nat Biotechnol 22, 1349

The global production of agricultural and food products is the world’s largest industry, with total revenues of ~$5 trillion (US) per year. Diseases caused by phytopathogens are economically important, resulting in multibillion-dollar losses annually, and it has been estimated that more than 25% of all crop plants worldwide are lost to fungal, bacterial, and viral diseases. In the developing world, up to 40% of all crop destruction can be directly attributed to plant diseases, with occasional catastrophic, life-threatening losses. The increased reliance on chemical solutions to diseases has compromised environmental quality, engendered a negative impact with consumers, and resulted in a rise of fungicide resistant microorganisms.

Conventional breeding, though relatively successful in the development of disease resistant plant cultivars, has become increasingly difficult because of the limitations of resistance genes within usable gene pools. However, advances in plant genetic engineering have facilitated the integration of beneficial "designer" genes into plants. This technology has been successfully applied to generate plant resistance to herbicides, some insects, and occasionally some phytopathogens. Strategies to improve the health of crops through genetic engineering have included transgenic expression of plant, fungal, or bacterial hydrolytic enzymes, pathogenesis-related proteins, components of plant defense-response pathways, antimicrobial proteins, and peptides. Although food safety is of utmost importance and disease resistant plants are required, the commercial application of this technology is lagging. The protection offered in some cases is limited to specific diseases, which means other pathogens can thrive and plants must still be sprayed with fungicides.

To address this problem, we have developed a platform methodology of protecting plants from a broad-spectrum of bacterial and fungal diseases. Work is in progress to extend this spectrum to viruses and insects. The technology is based on engineering antimicrobial peptides in plants. Because of their wide spectrum of antimicrobial activity and, at low concentrations, lack of toxicity to eukaryotic cells, the antimicrobial peptides represent promising candidates for transgenic application in plants.

Candidate antimicrobial peptides
Recently-discovered antimicrobial peptides are a class of potent, natural antibiotics found widely in nature from insects to plants and animals¹. Several related antimicrobial peptides have also been found in humans, where they are part of our early defense "innate" immune system. Depending on the source, these natural products vary greatly in their killing activity and spectrum, which extends from various types of bacteria to fungi, viruses, parasites, and even to such destructive pests as nematodes. The mechanism for killing microbial cells is multi-targeted and highly effective, and involves rapidly killing the microbe by drilling a molecular "hole" in its membrane, then inactivating and destroying its genome. Because the primary target of membrane-active, positively charged antimicrobial peptides is the cell membrane and not specific receptors or substrates, these peptides usually confer their activity against a broad spectrum of pathogenic microorganisms, and there is less probability of resistance arising by variation of its metabolic pathways.

Antimicrobial peptides of plant, insect, and amphibian origin have been expressed in transgenic plants, but only a few provide the target plants with any degree of broad-spectrum antimicrobial resistance. We have pioneered the development of unique, candidate target peptides (probiotics) with powerful broad-spectrum antimicrobial activities but with reduced cytotoxicity to plant and animal cells. This was achieved by using Insight II molecular modeling, and 2-D NMR structural determinations, combined with extensive screening against a variety of plant pathogens (collaboration: REW Hancock, UBC). These approaches have yielded three new target families of probiotics with fungal, bacterial, and viral disease resistance.

Shown below is a predicted model of temporin A and its modified analogue MsrA3². Using the Insight II (version 97.2) molecular modeling program, Homology (Molecular Simulations Inc., San Diego, USA), the temporin structure was drawn as an α-helix, based on the known α-helicity of temporin. The structure was then energy minimized using the Discover Program of Insight II.

Engineering select peptide / probiotics in plants
We used one of these peptides, MsrA1, to engineer broad-spectrum disease resistance in potato, including resistance to ‘late blight’, a disease of pandemic proportions². Potato is an ideal crop for the introduction of disease-resistance technology due to its severe and growing problems with bacterial, fungal, and viral diseases and accompanying storage losses.

The probiotic-enhanced potatoes demonstrated a phenomenal degree of broad-spectrum disease resistance to pathogens, including late blight and pink rot, and a variety of post-harvest pests. Potato tubers stored for over 27 months remain in nearly pristine condition when compared to unprotected tubers. These results have profound implications not only for diseases in the field but also with respect to the mitigation of storage crop losses (>$600B worldwide).

We have continued to test new peptides in potato and tobacco. Our recent data shows that MsrA3, a temporin analogue³, as well as MsrA2, derived from Dermaseptin⁴, and combinations thereof, provide a greater degree of disease resistance against a broad range of pathogens. When MsrA2 was used to inhibit the growth of agronomically important fungal pathogens, including Fusarium, Alternaria, Rhizoctonia, Phytophthora, and Pythium sp., its activity was far superior to the activities of other peptides tested. The estimated concentration of MsrA2 in leaf tissue was ~ 1 – 5 µg/g of fresh tissue, which, although not high, seems to be sufficient to protect the plants from the attack of pathogen(s)⁴. Constitutive expression of MsrA2 at this level is apparently non-toxic to transgenic plants, as no deleterious effects on the morphology or yield of plants and tubers could be seen.

A. Probiotic-expressing potato plant growing through mycelia of Fusarium sp.

B. Leaf derived from transgenic potato plant (right) expressing probiotic is resistant to P. infestans challenge. Leaf from control plant (left) is dead.

Regulated transgene expression, whereby a promoter is specifically activated in response to pathogen invasion or pest attack, has distinct advantages for genetic engineering disease/pest resistant traits in plants. We used a truncated 823 bp downstream fragment of the win3.12 promoter from poplar and showed that the win3.12 promoter-regulated expression of the antimicrobial peptide was sufficient to confer resistance against F. solani in transgenic tobacco.

This technology is considered a flexible platform, which can be extended to a host of economically important food and non-food crops. Equally important, plants can be used for cost-effective production of the peptides/probiotics destined for human pharmaceuticals and veterinary applications. We have extended this work to include canola, soybean, wheat, and even poplar, a model tree species.

Mycotoxins and food/feed safety
In addition to managing diseases, probiotic-enhanced plants can address the problem of food and feed safety. Phytopathogenic fungi not only cause a decrease in quantity and quality of crops, they often cause acute toxicity in animals and humans. Widely different genera of fungi (Fusarium, Penicillium, Aspergillus, Claviceps, Stachybotrys sp. etc.) produce mycotoxins in a wide variety of grains and foods. Mycotoxins are highly stable and cannot be destroyed by boiling, pressing, or processing, and infested produce has to be destroyed. Toxicological manifestations are both acute and chronic, such as cancer, immunosuppression, mutagenicity, and estrogenicity, and gastrointestinal, urogenital, vascular, renal, and nervous disorders. These mycotoxins can also be metabolized by animals fed contaminated grains and passed into milk, eggs, and other organs, thus reentering the food chain. Mycotoxin contamination is a worldwide problem affecting staple crops such as corn and small grains, as well as tree nuts, peanuts, sorghum, and many others. Maize, wheat, barley, and rice are high-risk commodities, and it is estimated that approximately 16,000 tons of maize, 123,000 tons of wheat and barley, and 12,000 tons of rice are affected by mycotoxins in southeast Asia alone. According to FAO estimates, world losses of foodstuffs due to mycotoxins are in the range of 1 B tons per year. One strategy to control mycotoxin levels is by controlling the growth of fungi.

We are testing the ability of our peptide probiotics to control Fusarium Head Blight (FHB) of wheat and barley caused by Fusarium graminearum. FHB has emerged as one of the most serious and damaging diseases of small grains. Trichothecenes are the virulence factors produced by the fungus. We are introducing probiotic genes into wheat in order to increase the FHB defense mechanism in wheat spikes and hence reduce or prevent the initial infection.

Summary
Our current growing reliance on antimicrobial chemicals remains unabated and is compromised due to the rise of fungicide and bactericide resistant microorganisms. We believe that the widespread incorporation of probiotic-enhanced plants and crops will make a large contribution to the general reduction in chemical solutions to disease control, increase yields, and reduce losses during storage. Furthermore, the antimicrobial peptides do not readily lead to microbial resistance and would help stem the rise of antibiotic and fungicide resistant microorganisms directly threatening human and animal health. The importance of feed or food microbial contamination in the industry is expected to increase with time. The emergence of pathogen strains resistant to conventional antibiotics and pesticides currently in use warrants application of new approaches to the containment of pathogenic microbes and to enhance food safety.

1. Hancock REW & Lehrer R (1998) Trends Biotechnol 16: 82–88

2. Osusky M et al. (2000) Nature Biotechnol 18: 1162–1166

3. Osusky M et al. (2004) Transgenic Research 13: 181-190

4. Osusky M et al. (2005) Theoretical & Applied Genetics DOI:10.1007/s00122005-2056-y

APPLICATION OF A REGENERATION QTL GENE TO PLANT TRANSFORMATION
Asuka Nishimura

Plant culture systems are vital to many areas of plant science and crop improvement, particularly in plant transformation. The ability of plants to regenerate is essential for establishing a successful plant culture system. However, not all plant species or varieties can regenerate easily. Generally, it is difficult to culture and regenerate agronomically important crops such as rice, wheat, and maize. In rice, an efficient culture system using mature seeds has been established for some research model varieties such as Nipponbare (Japonica) and Kasalath (Indica). Conversely, some leading varieties used for food production, such as Koshihikari (Japonica) in Japan and IR64 (Indica) in tropical countries, have low regeneration ability, which is a serious obstacle to production of transgenic plants.

The ability to regenerate is mainly controlled by quantitative trait loci (QTLs); however, no specific gene has yet been identified and the molecular mechanisms of regulation are not well understood. Therefore, the challenge is to identify optimal culture conditions required for regeneration in varieties with low regeneration success. A potential solution to this problem is to identify the QTL genes associated with regeneration ability, and to transfer the high regeneration ability QTL gene(s) into low regeneration varieties. However, this method is especially laborious and gives no understanding of the molecular mechanisms of plant regeneration. Recently, we succeeded in isolating a rice regeneration QTL gene using a map-based cloning method¹.

Isolation of a rice QTL gene in Japonica rice
We first chose Kasalath (Indica) as a high-regenerative variety to cross with a low-regenerative variety, Koshihikari (Japonica). The resulting Koshihikari x Kasalath F₁ plants were backcrossed with Koshihikari to produce 99 BC₁F₁ plants for which the genotypes of each plant were determined. Next, twenty BC₁F₂ seeds from each BC₁F₁ line were tested for regeneration ability and then subjected to QTL analysis. As a result, we found four putative QTLs located on chromosomes 1, 2, 3, and 6 in which Kasalath alleles were correlated with a positive effect on regeneration ability. The QTL located at around 45.4 cM on chromosome 1 had the largest effect and was designated Promoter of Shoot Regeneration 1 (PSR1). Fine mapping analysis of ca. 3,800 BC₃F₂ seeds revealed that PSR1 lies within a 50.8-kb interval between two molecular markers. In this region, four putative genes were predicted. Complementation analyses with several fragments covering each candidate gene indicated that the PSR1 gene may be a putative ferredoxin-nitrite reductase (NiR) gene.

We compared the NiR sequences of Koshihikari and Kasalath and found many polymorphisms, especially in the promoter regions. Therefore, we examined NiR expression in the callus using semi-quantitative RT-PCR. NiR expression was detected in both Koshihikari and Kasalath, but the expression level was much lower in Koshihikari. Immunoblot assays also showed that the NiR protein was expressed at a much higher level in Kasalath than in Koshihikari. Furthermore, enzymatic analyses revealed that NiR activity in extracts of Koshihikari calli was about 22-times lower than that of Kasalath. We also examined the relationship between the NiR activity in calli and regeneration ability in various kinds of Japonica rice varieties and found that the varieties with high regeneration ability always had high NiR activity, and the low regeneration varieties showed low NiR activity. This indicates that NiR activity is a determining factor for the regeneration ability of Japonica rice varieties.

NiR catalyzes the reduction of nitrite to ammonium and is a key enzyme in nitrate assimilation. Nitrate is commonly used as a nitrogen source for plant cell culture but its metabolite, nitrite, has a toxic effect on plant cell growth. Thus, rapid metabolism of nitrite is crucial for plant cell growth and regeneration. In this context, it is reasonable that Kasalath calli, having higher NiR activity, can rapidly metabolize nitrite while Koshihikari calli cannot. We measured the accumulation of nitrite in callus culture media and found an appreciable accumulation of nitrite ions in the Koshihikari culture medium, but we were not able to detect nitrite in the Kasalath medium. It is likely, then, that nitrite assimilation is a critical process for rice plant regeneration.

In Koshihikari, lower NiR activity was detected not only in calli, but also in normally grown root and leaf tissues. However, no growth defects were observed in Koshihikari. Therefore, it was thought that the nitrate concentration in the culture medium was not suitable for Koshihikari regeneration. We tried Koshihikari culture using several nitrogen conditions and could not find the best medium for Koshihikari regeneration.

As shown in these experiments, QTL gene isolation can be an efficient method for understanding regeneration mechanism(s) and finding optimum culture condition(s). In our preliminary studies, it was shown that the key genes regulating regeneration ability in Indica rice varieties were different from those in Japonica rice. An Indica variety such as IR64 has high NiR activity but low regeneration ability. We are now using QTL gene isolation to resolve the low regeneration ability in Indica rice using a strategy similar to the one we used with Japonica rice. We think these results in rice will help improve the regeneration ability of other crops and elucidate the molecular mechanisms of plant regeneration.

Application of NiR in transformation
Genes encoding antibiotic and herbicide resistance are widely used as selection markers in plant transformation. Recently, a positive selection system based on the E. coli phosphomannose isomerase (PMI) gene as a selection marker was developed in various plants, including rice². These selection markers are exogenous to plants. Moreover, several methods have been reported to generate selection marker-free transgenic plants, including site-specific recombination and intrachromosomal recombination to remove the selection marker, co-transformation, and transposable elements to segregate the selection marker³. A transformation method in chloroplasts has been established without using an antibiotic resistance gene⁴. However, these methods are generally time-consuming and inefficient. We hypothesized that the NiR gene we identified could be used as a selection marker for gene transformation because the introduction of the Kasalath NiR genome sequence conferred regeneration ability on Koshihikari. If the Kasalath NiR gene is co-delivered with the gene(s) of interest into Koshihikari, only successfully transformed cells will regenerate.

To test the efficiency of NiR as a selection marker, we constructed five vectors carrying the beta-glucuronidase gene (GUS) as a reporter:

• ü Kasalath NiR genome + 35S promoter::GUS (plasmid 1);

• ü Kasalath NiR promoter::NiR cDNA::NiR terminator + 35S promoter::GUS (plasmid 2);

• ü Rice Actin1 promoter::NiR cDNA:: NiR terminator + 35S promoter GUS (plasmid 3); and

• ü two control vectors not containing NiR (which only have the 35S promoter: plasmid 4, and an empty vector: plasmid 5).

These constructs were transformed into Koshihikari calli by Agrobacterium tumefaciens-mediated transformation by standard methods and cultured on a medium containing no chemicals for selection. Calli transformed with the three constructs carrying the Kasalath NiR (plasmid 1 – 3) formed many shoots that displayed GUS activity. On the other hand, calli transformed with constructs lacking Kasalath NiR (plasmid 4 and 5) formed either no shoots or a few non-transformed shoots. About 54% of calli transformed with Kasalath NiR as a selection marker regenerated into plants. Of these, more than 77% stained for GUS activity. These results indicate that the Kasalath NiR gene is useful as a selection marker for transformation of Koshihikari rice.

We also attempted to adopt the Kasalath NiR selection system for high regenerative varieties. Because nitrite is toxic to plants, the addition of excess nitrite in the culture medium causes growth inhibition of calli regardless of their regeneration ability. For example, the high regeneration varieties, Nipponbare and Kasalath, cannot grow under high-nitrite conditions. However, cells that were transformed with the NiR overexpression construct (plasmid 3) grew normally and showed GUS activity. This demonstrates that NiR can be used as a selection marker for transformation even in rice plants with high regeneration ability.

Future perspectives on new transgenic crops
A major advantage of using NiR for selection is that it is an endogenous rice gene that does not confer a selective advantage beyond that which may be found widely within the rice species. Unfortunately, it is likely that this NiR selection system cannot be applicable to all rice plants as described above. The regeneration ability of each rice plant would likely have been separately altered during evolution or the breeding process. However, we think that a similar selection system using endogenous genes is possible in any plant species having two varieties with differing regeneration ability—the QTL genes can be isolated and used in transformation.

We have been developing a high-throughput QTL isolation system in rice. By using this system, we aim to isolate various rice QTL genes, including regeneration genes, and hope to find a gene that is applicable to other crops such as maize and wheat. Moreover, we have been isolating some promoters for callus-specific expression of selective markers, which would be important for preventing marker expression after regeneration.

This expression control system could become standard for new transgenic crops. It would also be important to develop an efficient homologous recombination technique for removing the risks of unintentional effects, such as those caused by positional effect and copy number. Combining these systems would enable a transformation protocol that could ease some public concerns concerning genetically modified crops.

1. Nishimura A et al. (2005) Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems. Proc Nat Acad Sci 102, 11940-11944

2. Datta K et al. (2003) Bioengineered ‘golden’ indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnol J 1, 81-90

3. Ebinuma H et al. (2001) System for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20, 383-392

4. Daniell H et al. (2001) Antibiotic-free chloroplast genetic engineering: an environmentally friendly approach. Trends in Plant Science 6, 237-239

USING TRANSCRIPTIONAL ANALYSIS TO DETERMINE RESPONSES TO PHOSPHATE DEPRIVATION
M C Thibaud and L Nussaume

Phosphate (Pi) is an essential macronutriment required for plant growth and development. Its low availability to plants in many soils results not only from limiting amounts but also from its association with cations and organic compounds that create insoluble complexes. Thus, Pi has become one of the major plant nutrition problems limiting growth in both acidic and calcareous soils¹. Nevertheless, applications of large quantities of fertilizers to correct this problem are not economically sustainable and also lead to environmental pollution.

In most environmental conditions, plants must cope with limiting amounts of soluble Pi (5mM Pi compared to 500mM in controlled conditions). Significant changes in plant morphology and biochemical processes are associated with phosphate (Pi) deficiency^1,2 and result in plant adaptation to this abiotic stress (Fig. 1). However, the molecular bases of these responses to Pi deficiency are not thoroughly elucidated. Therefore, efforts have been made to understand the molecular basis of plants responses to Pi deficiency and to identify Pi-responsive genes whose expression can be manipulated to enable plant growth in low Pi environments.