Function Analysis Of Acid Phosphatase 1(OsACP1)in Pi Stress Adaptation Of Rice

Phosphorus is one of the essential elements for plant growth and development.It not only participates in the composition of DNA,RNA and phospholipid,but also plays an important role in the basic physiological metabolism processes,such as photosynthesis,respiration and energy metabolism.Plants can only absorb and utilize soluble inorganic phosphate(Pi),but most of the phosphorus in the soil exist in the form of organic phosphate or fixed inorganic phosphate.Therefore,the available Pi in the soil is usually not enough to support the plant growth and development.In order to adapte the lack of available Pi in soil,plants have evolved a series of responses to maintain their growth under Pi deficient condition,which include optimiazing root architecture,forming mycorrhizal symbionts,inducing high affinity phosphate transporters,secretion of organic acids and acid phosphatases.These responses could increase the absorption and utilization of Pi in plants.In addition to degrade organic phosphate from environment,intracellular acid phosphatases enhanced the recycling and re-utilizaiton organic phosphate in cell.In this study,we found that the expression of acid phosphatase 1(Os ACP1)was significantly induced by Pi deficiency and Os PHR2 through the analysis of rice transcriptome data,which suggested that Os ACP1 might be involved in the adaptive response of rice to Pi deficiency.According to bioinformatics analysis,Os ACP1 belongs to the halophilic dehalogenases(HAD)superfamily and have several homogures in different crops.Interestingly,these HAD homogure genes were reported to be involved in the regulation of Pi deficiency responses,though the molecular mechanism is unclear.In order to study the physiological and biochemical functions of Os ACP1,we first analyzed the spatiotemporal specific expression of Os ACP1 gene.RT-PCR analysis showed that the expression of Os ACP1 was specifically induced by Pi deficiency.Furthermore,real-time quantitative PCR and Os ACP1 promoter::GUS analysis demonstrated that Os ACP1 was significantly induced by Pi deficiency in both the aboveground and underground tissues.To further study the enzyme characteristics of Os ACP1,we constructed the prokaryotic expression vector of Os ACP1 by seamless cloning technology,and purified the protein of Os ACP1 by prokaryotic expression system.The results of enzyme activity and substrate specificity showed that the optimal p H of Os ACP1 protein was 6.0 and coenzyme ion was magnesium.Os ACP1 protein showed activity to a number of organic phosphate substrates,with the highest activity on phosphoethanolamine.Using rice protoplast transformation system,we found that the fusion protein of Os ACP1-GFP was mainly located in endoplasmic reticulum and Golgi membrane of rice cells.In order to analyze the function of Os ACP1 in the adaptability of rice to Pi deficiency,we cloned the target gene and created the overexpression and CRISPR mutants of Os ACP1 by Agrobacterium mediated transgenic technology.Compared with the wild type,the growth of rice plants with overexpression of Os ACP1 gene was significantly inhibited and the Pi content was significantly increased.The first leaf tip of 10 day old seedlings showed a Pi toxicity phenotype,which disappeared under Pi deficiency condition.Therefore,it is suggested that overexpression of Os ACP1 may affect the intracellular Pi balance and the growth of rice under normal growth condition.Under normal and Pi deficient conditions,the growth phenotype of the mutant was also inhibited compared with that of the wild type,but there was no significant change in leaf Pi content.The analysis agronomic characters in field further confirmed that the growth of overexpression and mutant of Os ACP1 were significantly inhibited.In conclusion,the physiological and biochemical functions of rice Os ACP1 gene were analyzed by a series of techniques such as molecular biology,physiology and biochemistry.It was preliminarily proved that Os ACP1 may participate in the reconstruction of endoplasmic reticulum and Golgi membrane phospholipids,recycling Pi from ethanolamine and choline phosphates,and maintain Pi homeostasis in rice under Pi deficient condition.