The Physiological Mechanism And Molecular Biology Of Phosphorus Acquisition In Rice (Oryza Sativa L.)

As one of indispensible macronutrients in plants, phosphorus acts as important components of nucleic acid, phospholipids, and ATP. It plays an important role on improving the utilization efficiency of phosphorus in soil and the fertilizers, by which to promote the sustainable development of agricultural production. In this study, the genetic differences and the corresponding physiological mechanism of low-Pi tolerance, the responsive differential-expressed genes to starvation-Pi, and the molecular characterization, expression patterns, and putative function of phosphate transporter genes OsPT2 and OsPT4 in rice have been explored and analyzed. The results were as follows:1. Using ten rice cultivars (Japonica type) to be the materials, the growth and phosphorus acquisition properties of the cultivars under various Pi levels were studied. It was found that there were dramatic differences on the accumulative P amount per plant among the cultivars under the deficient-Pi (20μM Pi) condition. The cultivars could be grouped in high-, mid- and low- type based on the Pi acquisition capabilities. There were significant and most significant correlations between dry weight per plant, total phosphorus content and accumulative P amount per plant, respectively, suggesting that the plant growth status under deficient-Pi was regulated by the plant Pi acquisition capability at high extent. In the meantime, the plant height, leaf area per plant were also shown to be a similar pattern with the accumulative P amount per plant, whereas the root numbers per plant, average root length, and root volume were not correlated with the Pi acquisition capability. Thus, the uptake amount of phosphorus under deficient-Pi condition was mainly affected by the Pi acquisition capability. The relative more amount of Pi was the important biological basis that the high-efficiency rice cultivar had improved plant growth traits when exposed to low-Pi.2. Using different Pi efficiency cultivars, including high-efficiency cultivars TP309 and Youzhi 8, mid-efficiency cultivars Kenyou 2000 and Xin 90-3, and low-efficiency cultivars 9618 and Zao 88-1, to be the materials, the photosynthetic characterization and the activities of cellular protection enzymes were studied. Under deficient-Pi condition, the photosynthetic rates (Pn) in the tested cultivars were gradually decreased with the growth process. But the Pn were the highest in the high-efficiency cultivars, then in the mid-efficiency cultivars, and were the lowest in the low-efficiency cultivars. The chlorophyll (Chl) concentration, soluble protein content, and superoxide dismutase (SOD) activity showed similar patterns with Pn in the tested cultivars, with higher values in the high-efficiency cultivars. But the malondialdehyde (MDA) content was shown the reverse pattern, with the highest in low-efficiency cultivars, then the mid-efficiency cultivars, and the lowest in the high-efficiency cultivars. There were not correlations between stomatol conductance (Gs) and transpiration rate (Tr) with the phosphorus use efficiency. There were significant positive correlations between Pn, Chl, soluble protein content, and SOD activity with accumulative P amount per plant, and significant negative correlation between MDA content and accumulative P amount per plant, suggesting that the above parameters could be used as the criteria for identification of rice genotypes with high capabilities of low-Pi tolerance. It is found that higher SOD activities play important roles on lessening the cellular over-oxidation degree under deficient-Pi condition, and to be further functional in sustaining relative normal running of the photosynthesis mechanism under low-Pi stress.3. Using cDNA-AFLP approach, the differential expressed genes (DEG) responding to low-Pi stress in cultivar TP309 were analyzed. In total 54 DEG were identified, including 22 with putative functions and 32 with unknown functions. The DEGs could be grouped into six functional classes, such as signal transduction, transcriptional regulation, amino acid synthesis, trafficking, stress responsive, and protein synthesis.The gene of 14-3-3-like protein (AF451190)was up-regulated by low-Pi, implying that it play roles in Pi acquisition by improving the H+ -pump activation. The induced expression of serine/threonine gene (AK100849) was possibly involved in the intracellular low-Pi signal transduction. The signal peptidase gene (AB066265) was presumed to be associated with the cleaving and processing of particular signal components located at the intracellular membrane, by which to mediate the low-Pi signal transduction. In this study, OsPTF1(AY238991), the previously identified Pi-starvation responsive gene, being classified into bHLH type transcription factor, was also shown to be low-Pi induced. Therefore, this gene should play roles on improving the plant Pi acquisition under deficient-Pi condition. We also identified a heat-shock transcription factor gene (AK065643), with the putative functions to be studied, was induced by low-Pi. The up-regulated expression pattern of HAK8 (AJ427977) indicated that the potassium uptake pattern in rice was also modified by low-Pi. In this study, in total 8 genes grouped into stress responsive were identified. But the functions of the genes were to be further explored. Therefore, the response in rice plants to low-Pi were involved in a complicate biological process, such as inter- or intra-cellular signal transduction, the transcriptional regulation of the related signal components genes and downstream genes, and the response at physiological, biochemical, and phenotypic levels in plants.4. The open reading frame of rice phosphate transporter gene OsPT2 was 1587 bp, encoding 528 amino acids. The molecular weight of OsPT2 was 57.84 kDa, having 12 conserved transmembrane domains and with an isoelectric point 8.68. Phylogenetic analysis indicated that OsPT2 had high similarities with phosphate transporter genes of rice OsPT1, PT1, PTs1 and OsPT3,wheat PT8, barley PT and maize PT4. Under normal Pi supply (CK,2 mM Pi) condition, the OsPT2 transcripts were detected in all roots and leaves, with a little higher amount in leaves. With the decrease of Pi, the expression level of OsPT2 in roots was elevated. But the expression level in leaves was not changed. Under low-Pi (20μM Pi) condition, the expression levels in roots were increased with the exposed time extension of low-Pi. A negative regulation effects on OsPT2 were found by NH4+, but no regulation effects on OsPT2 were identified by NO3-, K+, Fe2+ and Zn2+. Based PLACE analysis, some important regulatory elements related to stress responsive had been identified. One of them, named as PIBS and previously identified to be a low-Pi responsive element, was possibly involved in the elevation of the OsPT2 expression under deficient-Pi condition. In the transgenic tobacco plants in which the reporter gene Gus (β- Glucuronidase) was under the control of OsPT2 promoter, gave a similar histochemiscal staining results with the OsPT2 expression patterns in roots and leaves under various Pi-supply conditions.The transgenic plants fused the OsPT2 open reading frame were generated. Compared to the wild type plants (CK), the transgenic plants from the lines with varied OsPT2 expression levels, including line 1, 4 and 6, had not changed on the total phosphorus content. But the plant dry weight, accumulative P amount per plant, and photosynthetic rate (Pn) in transgenic plants were all higher than those in CK, showing a pattern of much more improvement on above parameters, with the increase of the OsPT2 transcripts. Therefore, ectopically expression of OsPT2 had a function on improving the Pi acquisition capability and photosynthesis in plants.5. The open reading frame of rice phosphate transporter gene OsPT4 was 1617 bp, encoding 538 amino acids. The molecular weight of OsPT4 was 58.83 kDa, having 11 conserved transmembrane domains and with an isoelectric point 8.24. Phylogenetic analysis indicated that OsPT4 had high similarities with rice Pht11-2, PT5, barley HvPT4 , wheat PT1, and maize PT2. Under various Pi supply (2 mM ~20μM Pi) condition, no OsPT4 transcripts were detected in roots. The expression levels in leaves were gradually increased with the decrease of Pi-supply amount. Under low-Pi (20μM Pi) condition, the expression levels in leaves were increased with the exposed time extension of low-Pi. No regulation effects on OsPT4 by the supply levels of NH4+, NO3-, K+, Fe2+ and Zn2+ were identified. Based PLACE analysis, some important regulatory elements related to stress responsive had been identified, but the low-Pi responsive element PIBS was not figured out, suggesting other regulatory elements being possibly involved in the OsPT4 responding to the low-Pi cue. Similar to OsPT2 promoter transgenic analysis, the transgenic tobacco plants in which the reporter gene Gus (β- Glucuronidase) was under the control of OsPT4 promoter, also gave a similar histochemiscal staining results with the OsPT4 expression patterns.The transgenic plants fused the OsPT4 open reading frame were generated. Compared to the wild type plants (CK), the transgenic plants from the lines with dramatic higher OsPT4 expression levels, including line 1, 5 and 7, had not changed on the accumulative P amount per plant, and a little lower total phosphorus contents. But the plant dry weight and photosynthetic rate (Pn) in transgenic plants were all higher than those in CK. In the meantime, the transgenic plants were all had higher phosphorus utilization efficiencies, the ratios of dry weight per plant to the accumulative P amount per plant, than CK under the deficient-Pi condition. Therefore, ectopically expression of OsPT4 had no functions on improving the plant Pi acquisition capability, but played roles on the transportation of phosphorus in the plants, by which to play roles on increasing the phosphorus utilization efficiency in plants under low-Pi stress condition.