Information Systems for Biotechnology

Plants have developed many physiological and biochemical systems of adaptation to Pi-deficiency stress. Thousands of genes, including many transcription factors, can simultaneously be regulated by Pi-deficiency stress in rice (2), indicating that plant adaptation to Pi-deficiency is systemic. Based on biochemical and transcriptional analysis, several adaptation systems have been revealed, including enhanced uptake ability through activation of high affinity transporters and adaptative root development; induction of phosphate scavenging and recycling enzymes; induction of alternative pathways of cytosolic glycolysis; induction of tonoplast H+-pumping pyrophosphatase; and alternative pathways of respiratory electron transport. The fact that many of the molecular and biochemical changes in response to Pi-deficiency occur in synchrony suggests that the genes involved are coordinately expressed and share a common regulatory system. A specific Pi-signaling pathway and regulation system in rice has been revealed (3), which makes it scientifically feasible to modify some key regulator(s) controlled by the specific Pi-signaling pathway to enhance the uptake and use efficiency of Pi through genetic engineering.

We found a transcription factor, with a Pi-deficiency responsive bHLH domain, in rice roots obtained from a cDNA library constructed by the Suppression Subtractive Hybridization (SSH) method. We cloned the gene, designated OsPTF1 (Oryza sative L. phosphate transcription factor), from an indica landrace rice variety Kasalath, which is Pi-deficiency tolerant. Using Agrobacterium-mediated transformation, we introduced the transcription factor into Oryza sativa cv. Nipponbare, which is sensitive to Pi-deficiency. Our experiments demonstrated that transgenic rice overexpressing OsPTF1 under the control of CaMV 35S showed enhanced tolerance to Pi-deficiency under both solution culture and soil experiments (4) (Figure 1). When grown in Pi-deficient conditions, the genetically modified (GM) rice plants produced longer roots and higher root biomass. About 30 percent more phosphate was absorbed by the GM rice plants than the wild type rice plants in the same environment.

To investigate the downstream genes regulated by OsPTF1, a microarray analysis was performed using rice whole genome oligo chips. Total RNA was extracted from 15d-old seedlings of the GM rice plants and the wild-type seedlings under Pi-supplied condition (10 mg Pi L-1). The sampling design is based on the fact that the plant PHO genes are induced only under Pi-deficiency condition-if the PHO genes can be induced under Pi-supplied conditions in the GM rice plants, then the overexpressed OsPTF1 should function in the induction. Quantitative PCR was used to confirm the differentially-expressed genes identified by the microarray analysis.

A high degree of concordance (r = 0.87) was observed between the results generated by the two methods. The microarray data showed that 158 genes, with a ratio greater than 2-fold in roots and/or in shoots, were found to be regulated by the overexpression of OsPTF1. The function-classified genes include nutrient transporters and metabolism, carbon metabolism, transcription factors, ATP-binding protein, oxidoreductase, protease, disease resistance protein, RNase, H+-transporting ATPase, vacuolar H+-pyrophosphatase, senescence-associated protein, receptor-like kinase, and several cytochrome P450 genes. Many "function unknown" or putative genes were strongly up- and down-regulated by overexpression of OsPTF1. Some of them did not respond to Pi-starvation (4). The remarkable induction of the PHO genes, like RNS1 and H+-transporting ATPase, in the GM rice plants under Pi-supplied condition strongly suggests that overexpression of OsPTF1 triggers a rescue system in response to Pi-starvation and plays a role in the increased tolerance to Pi-deficiency.

The bypass pathways, which replace Pi-requiring and adenylate-requiring enzymes using pyrophosphate-dependent and NADP-dependent enzymes, are considered important in maintaining carbon flux under Pi-starvation. Phosphoenolpyruvate (PEP) plays a central role in the modification of carbon and energy metabolism in response to Pi-starvation. In the cytosol, PEP can be converted to pyruvate catalyzed by pyruvate kinase (PK) or to oxaloacetate (OAA) catalyzed by PEP carboxylase (PEPC). The latter has been suggested to be a Pi-starvation induced bypass to preserve Pi. In this study, we did not find the gene for PEPC to be regulated, but a gene for PEP carboxykinase was up-regulated by Pi-starvation and overexpression of OsPTF1 in the shoots. PEP carboxykinase catalyzes the conversion of phosphate and OAA to PEP and CO2. The results suggest that the stimulation of recycling of the original metabolic pathways should be important in alleviating Pi-deficiency conditions, which was enhanced by the overexpression of OsPTF1.

Cloning of OsPTF1 may speed up the molecular breeding program for crops with enhanced tolerance to Pi-deficiency; because OsPTF1 was derived from rice rather than a different plant species, new rice varieties containing the modified gene could be developed by combining traditional breeding with molecular techniques. This study provides evidence that modification of a key regulator involved in the Pi-signaling pathway may exploit the potential ability of plants to more efficiently uptake Pi in growth medium and utilize Pi in plants.

Acknowledgements The author's research is supported by the Key Basic Research Special Foundation of China, Special Program of Rice Functional Genomics of China, National Education Ministry of China, and Science and Technology Bureau of Zhejiang province.

References

1. Vance C P et al. (2003) Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytologist 157, 423-447

2. Wasaki J et al. (2003) Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant, cell and Environment 26, 1515-1523

3. Hou XL et al. (2005) Regulation of the expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signaling and hormones. Plant Cell and Environment 28: 353-364

4. Yi KK et al. (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice (Oryza sativa L.). Plant Physiology, online