Evaluation Of Low Phosphate Tolerance At Seedling Stage And Molecular Essential Study On Phosphate Stress In Soybean

Phosphorus is one of the most important macronutrients required for plant growth and metabolism, and is the key component of nucleic acids, phospholipids and ATP as well as several enzymes and coenzymes. Phosphorus (in the form of phos-phate) deficiency is the second most frequently limiting macronutrient for plant growth mainly because it exists in the soil in complex, insoluble, inorganic and or-ganic forms that cannot be acquired directly by the plant. It is well established that normal plant growth and productivity are dependent on the availability of inorganic phosphate (Pi). However, in many natural and agricultural ecosystems, plants often face conditions in which availability and mobility of phosphate are at lower extremi-ties. To cope with phosphate limitation, plants have evolved tightly controlled mecha-nisms to maintain phosphate homeostasis, which include the development of lateral roots and root hairs, as well as more dramatic root structures such as proteoid and dauciform roots, the secretion from roots of phosphatases and organic acids, and the induction of high-affinity and some low-affinity Pi transporters (phosphate transporter, PT). Many plants also establish symbiotic associations with mycorrhizal fungi that aid Pi acquisition. A variety of adaptive strategies have evolved in plants that alleviate or help them cope with Pi deficiency. The implementation of these strategies requires changes in the expression profiles of hundreds of genes. The extent and complexity of the network of regulatory genes necessary to sense Pi status and regulate the deploy-ment of these adaptive strategies is now being revealed. The network components identified so far include transcription factors, SPX sub-family proteins, non-coding RNAs and protein modifiers, including proteins involved in SUMOylation, phos-phorylation, dephosphorylation and protein translocation.In this study, we cloned a number of related genes, such as PTs, PHRs (phos-phate starvation response), SPXs (Syg1, Pho81, XPR1) and Gm4s; studyed on struc-ture of GmPTl and GmPT2with bioinformatics; analysed subcellular localization of GmPT1and GmPT2with green fluorescent protein fused to the two PTs; studied on functional and biochemical analysis of GmPT1and GmPT2in yeast mutant; analysed expression pattern of GmPT1and GmPT2with Real-time RT-PCR; studied on the interaction of GmPHR1and GmSPX1; At the same times, we used the principal com-ponents and membership function analysis to evaluate low phosphate tolerance of20soybean genotypes. The results are as follows:1. We used the principal components and membership function analysis to evaluate low phosphate tolerance of soybean genotypes. The tolerance (from strong-est to weakest) as follows:Gantai, Wuheqihuangdou,7650, Xiangdou No.4, Xianjin No.2, Su88M-21, Peking, Gaozuoxuan No.1,Kefeng No.1, Xinyixiaoheidou, Nannong1138-2,94-156,87-23, He84-5, Bogao, RN-9, Tongshanbopijia, Wan82-178, Xiangqiudou No.2, Dianjiangzaohuangdou.2. We identified two P; transporter genes in soybean located on chromosomes Gm10(41,391,168-41,393,008) and Gm20(42,980,124-42,981,928). These genes are designated GmPT1(accession number HQ392508) and GmPT2(accessionnum-ber HQ392509), respectively. GmPT1is1841-bp long and contains an open reading frame encoding a536amino acid polypeptide (molecular mass58730.46Da). GmPT2is1802-bp long and contains an open reading frame encoding a536amino acid polypeptide (molecular mass58627.29Da). Interestingly, the open reading frame in both genes spans base pairs23-1633. These genes are88.7%similar in nu-cleotide sequence and97.9%similar in amino acid sequence. The two polypeptides share the greater degree of similarity with the characterized PT from Arabidopsis, tomato, potato and barrel clover (Medicago truncatula). GmPT1and GmPT2have a very high degree of identity with fungal PT from the mycorrhizal fungus Glomus versiforme (GvPT) and the budding yeast Saccharomyces cerevisiae (PHO84). GmPT1shows76%and61%and Gm?T2shows76%and63%amino acid sequence identity with GvPT (accession number Q00908) and PHO84(accession number P25297), respectively. Hydropathy plots of the deduced polypeptides suggest that GmPT1and GmPT2consist of12membrane-spanning regions, a feature shared by PTs, irrespective of the level of affinity. Computational modeling of the encoded proteins predicted a conserved secondary structure containing12transmembrane (TM) domains with a large hydrophilic loop between TM6and TM7.3. The TBpred Prediction Server was used for searches that yielded unambi-guous results with positive scores for the integral membrane protein. To verify the subcellular locations of GmPT1and GmPT2, a green fluorescent protein (GFP)-tagged gene was fused to the3’end of the open reading frame of the GmPT1or GmPT2genes. A clear GFP signal was observed at the periphery of onion epi-dermal cells bombarded with the GmPTl/GFP or GmPT2/GFP construction, whe-reas the signal was seen throughout cells expressing free GFP. Localization of the GmPTl/GFP and GmPT2/GFP fusion proteins to the periphery of the cells indicated that the two proteins are targeted to the plasma membrane. This is consistent with the results of earlier biochemical studies and together these data suggest that the GmPT1and GmPT2proteins are located in the plasma membrane.4. We used uptake studies with inhibitors to confirm the pH dependence of Pi transport. Pi transport activity was assessed at pH values in the range4-7. Differ-ences were detected in the activity profiles but the uptake rate was maximal at pH4and increased as the pH was reduced from7to4in each case. To investigate this in-fluence of a proton motive force on Pi transport activity, the uncouplers2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which destroy proton gradients across membranes, were applied. DNP at a concen-tration of100μM reduced the Pi uptake rate to79%(GmPT1) and82%(GmPT2) compared with100%uptake in the inhibitor-free control. The rate of uptake was re-duced to77%(GmPT1) and80%(GmPT2) by100μM CCCP and, to82%(GmPT1) and83%(GmPT2) by100μM Vanadate, an inhibitor of P-type H+-ATPases. The transporter rate was decreased significantly compared to that in the control. These results confirmed the hypothesis that Pi/H+cotransport via GmPT1and GmPT2de-pends on the pH gradient across the cell membrane that is maintained by the en-dogenous plasma membrane H+-ATPases. Moreover, competition studies showed that different anions did not reduce the Pi uptake rate, demonstrating the high degree of specificity of GmPT1and GmPT2for Pi. Strains carrying the GmPTl or GmPT2cDNA generally uptake Pi at rates similar to those of the vector controls at millimo-lar concentrations of Pi. In uptake experiments with radioactive Pi, the rate of trans-port was linear with time during the first5min of uptake under the conditions ap-plied. In three parallel experiments, the Lineweaver-Burk diagram, calculated using reciprocal uptake velocities at5min after addition of32Pi, indicated that Pi uptake facilitated by GmPT1and GmPT2followed Michaelis-Menten kinetics with an ap-parent Km valueof6.65mM and6.63mM, respectively. Thus, GmPTl and GmPT2are low-affinity Pi transporters that are dependent on the proton gradient across the plasma membrane.5. Root, stem and leaf tissues of7-day-old soybean seedlings were used to examine the expression of GmPT1and GmPT2. Expression of the two PT was en-hanced in both root and shoot during the first48h of Pi starvation. The expression of GmPTl and GmPT2in seedling tissues was increased during the3h after the Pi-sufficient treated seedlings were transferred to a P; deficient solution at48h com-pared to the expression measured in Pi-sufficient plants. The transcript levels of GmPT1and GmPT2were little changed in plants that were grown in half-strength nutrient solution for7days and then transferred to a Pi-sufficient solution. A de-crease in the transcript abundance of GmPT1and GmPT2in the leaf, stem and root of hydroponically grown soybean seedlings was apparent within3h of Pi deprivation. In conclusion, the expression level of GmPT1and GmPT2was not altered markedly and the change tendencies were complicated irrespective of how the seedlings were treated. Therefore, the soybean PTs GmPT1and GmPT2were slightly induced by P; deficiency.6. Interaction of GmPHRl with the promoter of GmSPXl was indicated by yeast one hybridization. The result indicated that GmPHR1can physically interact with GmSPXl through the cis-element, and this may be a key regulator in the Pi-signaling pathway in soybean.