Biochemical And Biological Functions Of Phosphate Starvation Responsive Protein Phosphatase,GmHAD1 Subfamily In Soybean

As one of the essential macronutrient element, phosphorus(P) plays a crucial role in plant growth and development. Due to its physical and biochemical properties, phosphate(Pi) is easily fixed by soils, wich results in low Pi avaibalility on soils. Low Pi availability becomes a limiting factor for crop growth and production on most of soils in China. Increased acid phosphatase(APase) activities are one of adaptive strategies for plants to P deficiency. Recently, haloacid dehalogenase(HAD) has been characterized as a novel APase. In this study, four GmHAD1 subfamily members were identified. Subsequently, their expression patterns, subcellular loccalization, biochemical properties were analyzed. Furthermore, functions of a Pi-starvation increased GmHAD1 member, GmHAD1-2 was analyzed. The main results were shown as follows:1. Alignment analysis of sequences from the four GmHAD1 subfamily members and their homologs from potato, common bean and Aarabidopsis, resulted in finding three unique and consreved motifs, including Dx Dx(T/V) at the N terminal(motif I), a conserved Ser residue(motif II), and K-(x) 18-22(GDxxx D) at the C terminal(motif III).2. Quantitative real time PCR(q PCR) was conducted to examine expression patterns of four GmHAD1 members in two soybean genotypes, BD2(a P-inefficient genotype) and BX10(a P-efficient genotype). Results showed that expression levels of four GmHAD1 members were enhanced by Pi starvation in soybean. Among them, transcripts of GmHAD1-2, GmHAD1-4 and GmHAD1-7 were increased by more than tenfolds in soybean roots and leaves, transcripts of GmHAD1-6 were increased by more than twofolds.3. Through tobacoo leaf epidermal cell transient expresison systerm, subcellular localzaition of four GmHAD1 members was analyzsed. Results showed that GmHAD1-2, GmHAD1-4, GmHAD1-6 and GmHAD1-7 located in both nucleus and plasma membrane.For enzymatical analysis, recombinant GmHAD1-2, GmHAD1-4, GmHAD1-6 and GmHAD1-7 fused with glutathione-S-transferase(GST) was purified from E.coli. It was shown that GmHAD1-2, GmHAD1-4, and GmHAD1-7 possessed the strongest activities toward phosphorylated serine, respectively. However, GmHAD1-6 exhibited the highest activities against phosphorylated tyrosine.4. Expression patterns of GmHAD1-2 were futher investigated through histochemical analysis of Arabidopsis transformed witht GmHAD1-2 promoter fusion glucuronidase(GUS). The result showed that low Pi avalibility enhanced expression levels of GmHAD1-2 in leaves and vascular tissues of roots.5. Biological functions of GmHAD1-2 were futher investigated in soybean composite plants with overexpressing or suppressing GmHAD1-2. Resutls showed that suppressing GmHAD1-2 resulted in a significant decrease of biomass, P content, f taproot and total root length, strongly suggesting that GmHAD1-2 could regulate root growth and Pi accumulation.