Functional Analysis Of A Root Specific Phosphate Transporter,GmPT4, In Soybean

Phosphorus(P)is one of the essential mineral elements for plant growth and development. Despite the total content of P is relatively high in most soil, P is easily fixed by soil particles. So the availability of P is very low, and it is hard to be absorbed directly by plants. Soybean(Glycine max)is a common economical crop, but low P availability restricts the growth and productivity of soybean. The Gm Pht1 gene family is assumed to be the predominant system responsible for Pi uptake by soybean roots. Therefore, for improving the phosphorus uptake efficiency, the study of soybean transporter Gm PTs is particularly important. In this study, we firstly compared the phosphorus efficiency between two soybean genotypes, genome sequencing variety Williams82 and P-efficient soybean variety YC03-3 breeded by our university. Then, we analyzed the responses of Gm PTs to the changes of Pi concentration, and screened out the root-specific Gm PTs which is sensitive to phosphorus. After that, we further analyzed the post-transcriptional regulatory mechanism of Gm PTs phosphorylation. Based on the above results, we studied the screened Gm PT4 via physiological and molecular experiment, and further investigated its functions in soybean's adaptation to LP conditions. The main results were shown as follows:1) Low P significantly inhibited the growth of soybean under hydroponic conditions.Compared to the normal condition, low P significantly inhibited plant dry weight and root growth. With treatment time prolonged, the inhibition level increased. There were significant genotypic differences between two studied soybean genotypes, and YC03-3performed better than Williams82 under LP conditions.2) Responses of Gm Pht1 gene family to the changes of Pi concentration showed that most of the soybean phosphate transporters had different responses to external P concentration changes. Among them, Gm PT1 had the highest relative expression level,followed by Gm PT4, and the expression levels of other Gm PTs were relatively low. The expression levels of Gm PT7 and Gm PT13 had fluctuations with the shift of day and night,suggested that the changes may be related to the circadian change. Moreover, the expression of Gm PT4 was induced rapidly under P deficiency, then reached the maximum after P-deficience for 36 h. After re-supplement of P, the expression of Gm PT4 decreasedrapidly. The expression of Gm PT4 changed with external P concentration, indicating that it might be the root specific sensitive phosphate transporter responing to the external P concentration change.3) The expression of Gm PTs in different root tissue under LP and HP conditions showed that Gm PT1, Gm PT4, Gm PT7, Gm PT12 and Gm PT13 mainly expressed in lateral roots under LP conditions. Gm PT2, Gm PT3 and Gm PT6 mainly expressed in lateral roots and taproots under LP conditions. Gm PT5 mainly expressed in taproots under LP conditions. The expression of Gm PT14 was relatively high in lateral roots under HP conditions. The expression of Gm PT8, Gm PT9 and Gm PT10 showed no difference between treatments.4) Bioinformatic analysis of candidate gene Gm PT4 showed that it contained 12 conserved transmembrane domains, and had 1 P1 BS and 3 W-box motifs in its promotor region. Phosphorylation sites prediction analysis indicated that it contained 19 potential phosphorylation sites, and the Ser-509 and Ser-514 had the highest scores. Subcellular localization demonstrated that Gm PT4 localized on the plasma membrane. Histochemical analysis showed that under HP conditions, Gm PT4 mainly expressed in stele, while under LP conditions, Gm PT4 was abundantly expressed in the epidermis, cortex and stele.5) Staining test showed that Gm PT4 and its point-mutant can partly restore Pi uptake activity in a yeast mutant MB192, which is defective in high-affinity Pi uptake. The substitution of Ser-509 to Ala or Asp in Gm PT4 did not impede its localization and function.The substitution of Ser-514 to Ala did not influence its localization, but conferred a decreased Pi transport capacity in yeast,indicating that Ser-514 plays an important role in the function of Gm PT4.6) Overexpression of Gm PT4 in transgenic soybean composite plants showed that Gm PT4 could increase the soybean biomass and P content, and RNA interference of Gm PT4 could decrease the soybean biomass and P content, suggested that Gm PT4 had important functions in mediating soybean Pi uptake.