Studies On Biological Functions Of TaPHT2;1and TaPT2, Two Phosphate Transporter Genes In Wheat (Triticum Aestivum L.), In Mediating Phosphorus Acquisition And Utilization In Plants

The phosphate transporters in plants play critical roles in mediating Pi uptake and thecellular translocation of Pi across cells and the tissues. In this study, in regard to fewinvestigations on cloning, molecular characterization analysis, biochemical propertyidentification, as well as biological functions of the phosphate transporter (PT) genes inwheat (Triticum aestivum L.), we have characterized two wheat PT genes, denoted asTaPHT2;1and TaPT2, respectively, based on sequencing and further homology analyzingthe clones of cDNA subtractive suppression hybridization (SSH) libraries enrichingsufficient Pi in leaves and deprivation Pi in roots. By using modern molecular biologyapproaches, the molecular characterizations and their roles in regulating plant phosphorusnutrition have been investigated. The main results are as follows:1. TaPHT2;1shares high homology with a subset of PHT2genes in diverse plantspecies. It is suggested to be a member of PHT2family. TaPHT2;1has a cDNA full lengthof2094bp which contains an open reading frame of1707bp and encodes a557aapolypeptide. TaPHT2;1contains twelve conserved spanning-transmembrane domains. Along hydrophilic region is located at the zone between domain7and domain8.2. Expression analysis revealed that TaPHT2;1was strongly expressed in the leaves,and was up-regulated by low Pi stress. The expression levels of TaPHT;1in roots weresignificantly lower than leaves under the conditions of Pi sufficience and Pi deprivation.Moreover, the expression of TaPHT2;1also exhibited a circadian rhythmic expressionpattern. During the photo phase progression, the expression of TaPHT2;1was graduallyincreasing whereas the TaPHT2;1expression was decreasing by degrees along with the progression in the dark phase.3. A TaPHT2;1–green fluorescent protein fusion (TaPHT2;1-GFP) was constructedand subjected to genetic transformation of tobacco and wheat. At the subcellular level, thefusions in the leaves of tobacco and wheat were detected specifically in the chloroplastenvelop. Using the genetic transformation approach, the ORF of TaPHT2;1was used totransform a yeast mutant that losing the function of high-affinity phosphate transportersystem. It was confirmed that TaPHT2;1could complement the Pi transporter activities inthe yeast mutant with a defect in Pi uptake. These results indicate that TaPHT2;1is locatedonto the chloroplast envelop in which it exerts the roles in mediating the Pi translocationbetween the cytoplasm and the chloroplast.4. Using the callus derived from wheat young embryos as the transformation explants,the expression cassette harboring the ORF of TaPHT2;1with anti-sense orientation wassubjected to transformation in wheat. A sub set of transgenic lines with down-regulation ofTaPHT2;1were generated. Down-regulated expression of TaPHT2;1significantly reducedPi concentration in the chloroplast under sufficient (2mmol/L Pi) and deficient Pi (100μmol/L Pi) conditions, suggesting that TaPHT2;1is crucial in the mediation of Pitranslocation from the cytosol to the chloroplast. The down-regulated expression ofTaPHT2;1resulted in reduced photosynthetic capacities, total P contents, and accumulatedP amounts in plants under sufficient and deficient Pi conditions, eventually leading toworse plant growth phenotypes.5. The TaPHT2;1knockdown plants exhibited pronounced decrease in accumulatedphosphorus in sufficient and deficient Pi conditions, suggesting that TaPHT2;1is animportant factor to associate with a distinct P signaling that up-regulates other PT membersto control Pi acquisition and translocation within plants. Therefore, TaPHT2;1is a keymember of the Pht2family involved in Pi translocation.6. TaPT2is highly similar to numerous PHT1family genes at the nucleic acid level. Itis belonging to the member of PHT1family. TaPT2is1802bp long at the cDNA level; itencodes a525amino acid polypeptide with a molecular weight of57.5kDa and anisoelectric point of8.65. Transmembrane prediction analysis suggested that TaPT2contains12conserved membrane-spanning domains and is transported to the cytosolicmembrane after endoplasmic reticulum sorting. 7. The open reading frame (ORF) of TaPT2was PCR amplified and integrated into ayeast expression vector which was further transformed into the cells of MB192, a yeastmutation that is losing of the high-affinity Pi transportation system. Functionalcomplement analysis revealed that TaPT2endowed Pi transporter activities in a yeastmutant that is defective in Pi uptake, with high-affinity Pi acquisition.8. TaPT2transcripts were specifically detected in the roots. The transcripts wereupregulated under Pi deprivation and downregulated under Pi sufficiency. These resultssuggest that TaPT2expression is associated with external Pi concentration. Using DNArecombinant technique, the expression cassettes harboring the TaPT2ORFs with sense-and antisense-orientations were constructed. Furthermore, the transgenic wheat plantstransformed the aforementioned expression cassettes were established by adopting theAgrobacterium tumefaciens-mediated transgene approach.9. Transgene analysis revealed that TaPT2overexpression or knockdown did notregulate plant dry mass production, Pi acquisition, and photosynthetic capacity under Pisufficiency. The effects of TaPT2on plant tolerating Pi deprivation were determined byhydroponically culturing wild type (WT) and the transgenic plants with sufficient-Pi anddeprivation-Pi nutrient solutions. Under Pi sufficiency, TaPT2overexpression orknockdown did not regulate plant dry mass production, Pi acquisition, and photosyntheticcapacity. Under Pi deprivation, TaPT2overexpression increased plant dry massaccumulation, total P content per plant, and photosynthetic efficiencies, whereas TaPT2downregulation reduced plant dry mass, accumulative P amount, and photosyntheticparameters. These results collectively suggest that TaPT2is a high-affinity PHT1memberthat has important functions in mediating plant Pi uptake under Pi deprivation.10. The results in this study indicate that TaPHT2;1and TaPT2act as the familymembers of PHT2and PHT1, respectively. They separately exert the roles in mediating Pitranslocation from the cytoplasm to chloroplast and in taking up Pi from the media underdeprivation Pi in plants. These genes have potential use in evaluating P use efficiency ofwheat genetic resources and in generating novel wheat germplasms (cultivars) with high Pusage capacity in the future.