The Discovery And Verification Of Phosphorus Efficient Utilization Related Gene In Rice Roots

Phosphorus is one of the major elements essentially for the growth and development of plants and also the lowest absorption efficiency element by plant. The rice yield increase is always dependent on the phosphate fertilizer application, which in turn lead to the eutrophication of waters, worsen environmental pollution, increase the production cost of agriculture and speed up the depletion of un-renewable phosphate resources. Therefore, to activate and absorb the fixed phosphorus in soil efficiently by plant itself and to reduce the dependence on phosphate fertilizer in agricultural practice is of great importance. In this paper, low-phosphorus (low-P)-tolerant rice variety Yi 2434 (YI 2434) and low-P sensitive variety Tongjing 981 (TJ 981) were employed as experimental materials to study the influence of low-P stress on the morphology, physiology and biochemistry in rice roots, and gene chip, qRT-PCR, proteomics technology used to detect the genes expression to discover phosphorus starvation signal transduction, phosphorus activation and efficient absorption, phosphorus metabolism and utilization related genes and verify the role of phosphorus efficient utilization related gene in rice roots undder the low-P stress.1. The effects of low-P stress on the morphology as well as physiological and biochemical characteristics in rice rootsAfter low-P treatment for 15 days, the root length, surface area, projected area and volume of root systems of both rice varieties were increased to some extent compared with those of control group, and low-P tolerant rice variety Yi 2434 showed more significant increase. The results suggest that the YI 2434 could increase the contact area between the rice root and soil, promote root ability of phosphorus absorption. Compared with control group, under low-P stress, the activity of both acid phosphatase (APA) in the tissues of roots and that secreted by roots in two rice varieties were increased. However, the APA activity of YI 2434 increased more significantly than that in TJ981,which could efficiently activate organic phosphorus in the plant and in the soil and consequently confer upon YI2434 higher tolerance ability to the low-P stress.2. The effects of low-P stress on the gene transcription level in rice rootsThe response of gene expression to low-P stress in Yi 2434 after 15d of low-P treatment was investigated by using gene chip.The results show that there are 1329 up-regulated genes(FC≥2.0) and 1987 down-regulated genes(FC< 0.5) induced by low-P stress and we mainly explored the effect and mechanism of the genes expression induced by low-P stress (p-value≤0.05, FC≥2.0) on the efficient absorption and utilization of phosphorus,including the genes related to phosphorus starvation signal transduction, phosphorus activation and efficient absorption, phosphorus metabolism and utilization.The results showed that, (1) the induced expression of phosphate starvation regulator protein 1(PHR1), miR399 and SPX genes which contribute to activating the phosphorus starvation signal pathway; (2) the induced expression of acid phosphatase (VPA),phosphoenolpyruvate carboxylase (PEPC), voltage-dependent anion channell (VDAC1), C4-dicarboxylate transporter/malic acid transport protein (C4-DT/MAT), phosphate transporter (OsPT2, OsPT6) and major facilitator superfamily protein (MFS) genes which contribute to the activation and high efficient absorption of phosphorus in rice roots; (3) the induced expression of nucleotide pyrophosphatase/phosphodiesterase (NPP), glycerophosphory diester phosphodisterase (GDPD), phospholipase (PLC, PLA2),inorganic pyrophosphatase(PPase), trehalose-6-phosphatephosphatase (TPP), Mannose-6-phosphate isomerase (MPI), protein-tyrosine phosphatase (PTP)and amino alcohol transferase 1 (AAPT1) genes which contribute to the metabolism and efficient utilization of phosphorus in rice.3. Determination of the key genes related to low-P stress in rice roots by qRT-PCRqRT-PCR results of gene transcription level after low-P treatment for different days show that, the expression of all the three groups of genes related to phosphorus starvation signal, phosphorus activation and high efficiency absorption as well as phosphorus metabolism and utilization in low-P tolerant rice variety YI 2434 and low-P sensitive variety Tongjing 981 were consistent with the results from gene chip,while the gene expression level in YI 2434 roots is obviously higher than that in TJ 981. The expression levels of 10 genes in both YI 2434 and TJ 981 were increased gradually with extension of low-P treated time and the increase amplitude of YI 2434 is significantly higher than that of TJ 981. Results suggest that the low phosphorus resistance of rice attribute greatly to the expression level of genes related to the phosphorus activation and highly efficient phosphorus absorption as well as phosphorus metabolism and efficient utilization.4. Proteomic change in rice root under the low-P stressMass spectrometry detect results show that quite a few protein expression levels in YI 2434 roots greatly increased after low-P treatment by using Two-dimensional electrophoresis and mass spectrometry detection technology, including the up-regulated expression of cytoplasmic malate dehydrogenase (cMDH) which maybe promote the secretion of organic acids and activated the organic phosphorus in soil;the up-regulated expression of adenosine kinase (AK), fructose diphosphate aldolase(ALD),phosphoglycerate mutase (PGAM) and enolase which regulate metabolic pathways and improved the utilization efficiency of phosphorus;the up-regulated expression of heat shock 70 kD protein (HSP70), Glutathione-S-transferase(GST) and L-ascorbate-Peroxidase-2 (APX2) enhanced the antioxidant ability of YI 2434 rice roots. The functions of protein differential expressions are partly consistent with the results of gene chip, indicating that the genes related to phosphorus highly-efficient absorption and utilization play essential role under low-P stress in plant.5.Major conclusionsCompared with TJ 981, the phosphorus content in the leaves of YI2434 showed a less decrease under low-P stress, indicating that Y12434 has the capability of efficient phosphorus absorption from low phosphorus medium to ensure the basic phosphorus demand of growth and development. The induced expression of PHR1, miR399 and SPX genes could regulate the expression of phosphorus activation and efficient absorption genes in the phosphorus signaling pathways in YI 2434 roots. The induced expression of APA, PAPs, PEPC, ADVC1, OsPT2 and OsPT6 and the up-regulated expression of cMDH proteins could accelerate the hydrolysis of organic phosphorus and activate the inorganic phosphorus and facilitate the phosphorus absorption. The induced expression of PPase, TPP, PLC,PLA2,NPP,GDPDD,PTP and the up-regulated expression of AK、ALD、PGAM proteins could optimize the phosphorus redistribution and utilization in rice.