Functional Analysis Of Genes In Rice Purple Acid Phosphatase Ia Subgroup

Phosphorous (P) is one of the important macronutrient nutrient required for plant growth. Plants take up P exclusively in the form of inorganic phosphate (Pi). To meet the needs for crop production, chemical fertilizer have been extensively used. The excessive use of fertilizers not only increase the cost of crop production, but also lead to severe water pollution. On the other hand, applications of chemical P fertilizers has created a huge P pool in soil, although the soil-fixed P is normally not absorbable for plants. To sustain our agriculture production system, it is an urgent task to genetically improve crop plants for high utilization efficiency of soil P. Under Pi deficient conditions, plants could enhance the P uptake through root morphological modification, activation of phosphate (Pi) transporters, alteration of inner P metabolism, improvement of available P in soil through secretion of purple acid phosphatases (PAP) into the rhizosphere. The secretion of PAP from plant roots could facilitate the conversion of organic P into Pi, thus alleviate plant Pi starvation status. In this study, rice PAPs, OsPAP10a and OsPAP10c, that belonged to PAP la subgroup were studied for their expression and function to assess the value of the two genes for genetic improvement of Pi use efficiency.Quantitative reverse transcription PCR (qRT-PCR) showed that expression of OsPAP10a in shoots and roots were induced specifically by Pi-deficiency whereas that of OsPAP10c was induced in roots but not in the shoots. Using genetic materials of OsPHR2-overexpression lines, pho2mutants and OsSPX1RNA interference lines, it was confirmed that the expression of OsPAP10a and OsPAP10c at both transcript and protein levels were regulated by PHR2-directed Pi signal pathway. In gel phosphatase activity assays of root protein extracts showed that there were three major acid phosphatase isoforms. El band with the slowest mobility showed significant induction by Pi starvation treatment. Mass spectrometry analysis showed that the El band contains both OsPAP10a and OsPAP10c.The full length cDNA of OsPAP10a was overexpressed in rice using Agrobacterium-mediated rice transformation. Two independent transgenic lines were produced, namely lOa-Oel and10a-Oe2Overexpression of OsPAP10a in a constitutive manner led to significant increase of phophatase activity in shoots and roots. Results showed that the root-associated acid phosphatase activity in10a-Oe1and10a-Oe2was significantly higher compared to that in the wild type plants. BCIP staining assay showed that OsPAP10a is a secreted protein, expressing in the root surface. When0.5mM ATP was used as the only external P source, the secreted PAP activity in the roots of transgenic plants could hydrolyze ATP to release Pi, which resulted in the significant increase of Pi concentration in the hydroponic medium grown the transgenic plants. In a pot experiment, two OsPAP10a overexpression lines showed increased tiller numbers compared to the control plants.Analysis of acid phosphatase activity and BCIP staining of the transgenic rice plants overexpressing OsPAP10c confirmed that OsPAP10c was also a secreted PAP, similar to OsPAP10a. Compard to the OsPAP10a, OsPAP10c has stronger enzyme activity than that of the OsPAP10a.In summary, our study showed that induced by Pi starvation, OsPAP10a and10c are two important acid phosphatases highly expressed in rice roots. Both of them exhibited great potential to be used in genetic improvement of rice Pi use efficiency.