Mian Gu

The Phosphate Transporter Gene OsPht1;8 Is Involved in Phosphate Homeostasis in Rice

Plant phosphate transporters (PTs) are active in the uptake of inorganic phosphate (Pi) from the soil and its translocation within the plant. Here, we report on the biological properties and physiological roles of OsPht1;8 (OsPT8), one of the PTs belonging to the Pht1 family in rice (Oryza sativa). Expression of a β-glucuronidase and green fluorescent protein reporter gene driven by the OsPT8 promoter showed that OsPT8 is expressed in various tissue organs from roots to seeds independent of Pi supply. OsPT8 was able to complement a yeast Pi-uptake mutant and increase Pi accumulation of Xenopus laevis oocytes when supplied with micromolar 33Pi concentrations at their external solution, indicating that it has a high affinity for Pi transport. Overexpression of OsPT8 resulted in excessive Pi in both roots and shoots and Pi toxic symptoms under the high-Pi supply condition. In contrast, knockdown of OsPT8 by RNA interference decreased Pi uptake and plant growth under both high- and low-Pi conditions. Moreover, OsPT8 suppression resulted in an increase of phosphorus content in the panicle axis and in a decrease of phosphorus content in unfilled grain hulls, accompanied by lower seed-setting rate. Altogether, our data suggest that OsPT8 is involved in Pi homeostasis in rice and is critical for plant growth and development.

A Constitutive Expressed Phosphate Transporter, OsPht1;1, Modulates Phosphate Uptake and Translocation in Phosphate-Replete Rice

A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, 33P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions.

Expression analysis suggests potential roles of microRNAs for phosphate and arbuscular mycorrhizal signaling in Solanum lycopersicum

MicroRNAs (miRNAs) have emerged as a class of gene expression regulators that play crucial roles in many biological processes. Recently, several reports have revealed that micoRNAs participate in regulation of symbiotic interaction between plants and nitrogen-fixing rhizobia bacteria. However, the role of miRNAs in another type of plant-microbe interaction, arbuscular mycorrhizal (AM) symbiosis, has not been documented. We carried out a microarray screen and poly(A)-tailed reverse transcriptase-polymerase chain reaction (RT-PCR) validation for miRNA expression in tomato (Solanum lycopersicum) under varying phosphate (Pi) availability and AM symbiosis conditions. In roots, miRNA158, miRNA862-3p, miRNA319, miRNA394 and miR399 were differentially regulated under three different treatments, Pi sufficient (+P ), Pi deficient (-P) and AM symbiosis (+M ). In leaves, up to 14 miRNAs were up- or down-regulated under either or both of the Pi treatments and AM symbiosis, of which miR158, miR319 and miR399 were responsive to the treatments in both roots and leaves. We detected that miR395, miR779.1, miR840 and miR867 in leaves were specifically responsive to AM symbiosis, which is independent of Pi availability, whereas miR398 in leaves and miR399 in both roots and leaves were Pi starvation induced. Furthermore, miR158 in roots as well as miR837-3p in leaves were responsive to both Pi deprivation and AM colonization. In contrast, miR862-3p in roots was responsive to Pi nutrition, but not to AM symbiosis. Moreover, the group of miRNA consisting miR319 and miR394 in roots and miR158, miR169g*, miR172, miR172b*, miR319, miR771 and miR775 in leaves were up- and down-regulated by Pi starvation, respectively. The data suggest that altered expression of distinct groups of miRNA is an essential component of Pi starvation-induced responses and AM symbiosis.

Identification of two conserved cis‐acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza‐activated phosphate transporters in eudicot species

• In this study, six putative promoter regions of phosphate transporter Pht1;3, Pht1;4 and Pht1;5 genes were isolated from eggplant and tobacco using the inverse polymerase chain reaction (iPCR). The isolated sequences show evolutionary conservation and divergence within/between the two groups of Pht1;3 and Pht1;4/Pht1;5. • Histochemical analyses showed that all six promoter fragments were sufficient to drive β-glucuronidase (GUS) expression specifically in arbuscular mycorrhizal (AM) tobacco roots and were confined to distinct cells containing AM fungal structures (arbuscules or intracellular hyphae). • A series of promoter truncation and mutation analyses combined with phylogenetic footprinting of these promoters revealed that at least two cis-regulatory elements--the mycorrhiza transcription factor binding sequence (MYCS) first identified in this study and P1BS--mediated the transcriptional activation of the AM-mediated inorganic phosphate (Pi) transporter genes. Deletion or partial mutation of either of the two motifs in the promoters could cause a remarkable decrease, or even complete absence, of the promoter activity. • Our results propose that uptake of inorganic phosphate (Pi) by AM fungi is regulated, at least partially, in an MYCS- and P1BS-dependent manner in eudicot species. Our finding offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi signalling pathways.

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.

Functional Characterization of 14 Pht1 Family Genes in Yeast and Their Expressions in Response to Nutrient Starvation in Soybean

Background Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide. Principal Findings We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean. Conclusions The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.

Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of Pht1 family genes in response to Pi deficiency in tomato

BackgroundPhosphorus (P) deficiency is one of the major nutrient stresses limiting plant growth. The uptake of P by plants is well considered to be mediated by a number of high-affinity phosphate (Pi) transporters belonging to the Pht1 family. Although the Pht1 genes have been extensively identified in several plant species, there is a lack of systematic analysis of the Pht1 gene family in any solanaceous species thus far.ResultsHere, we report the genome-wide analysis, phylogenetic evolution and expression patterns of the Pht1 genes in tomato (Solanum lycopersicum). A total of eight putative Pht1 genes (LePT1 to 8), distributed on three chromosomes (3, 6 and 9), were identified through extensive searches of the released tomato genome sequence database. Chromosomal organization and phylogenetic tree analysis suggested that the six Pht1 paralogues, LePT1/3, LePT2/6 and LePT4/5, which were assigned into three pairs with very close physical distance, were produced from recent tandem duplication events that occurred after Solanaceae splitting with other dicot families. Expression analysis of these Pht1 members revealed that except LePT8, of which the transcript was undetectable in all tissues, the other seven paralogues showed differential but partial-overlapping expression patterns. LePT1 and LePT7 were ubiquitously expressed in all tissues examined, and their transcripts were induced abundantly in response to Pi starvation; LePT2 and LePT6, the two paralogues harboring identical coding sequence, were predominantly expressed in Pi-deficient roots; LePT3, LePT4 and LePT5 were strongly activated in the roots colonized by arbuscular mycorrhizal fungi under low-P, but not high-P condition. Histochemical analysis revealed that a 1250-bp LePT3 promoter fragment and a 471-bp LePT5 promoter fragment containing the two elements, MYCS and P1BS, were sufficient to direct the GUS reporter expression in mycorrhizal roots and were limited to distinct cells harboring AM fungal structures. Additionally, the four paralogues, LePT1, LePT2, LePT6 and LePT7, were very significantly down-regulated in the mycorrhizal roots under low Pi supply condition.ConclusionsThe results obtained from this study provide new insights into the evolutionary expansion, functional divergence and genetic redundancy of the Pht1 genes in response to Pi deficiency and mycorrhizal symbiosis in tomato.

Phosphate transporter OsPht1;8 in rice plays an important role in phosphorus redistribution from source to sink organs and allocation between embryo and endosperm of seeds.

Phosphorus (P) redistribution from source to sink organs within plant is required for optimizing growth and development under P deficient condition. In this study, we knocked down expression of a phosphate transporter gene OsPht1;8 (OsPT8) selectively in shoot and/or in seed endosperm by RNA-interference using RISBZ1 and GluB-1 promoter (designate these transgenic lines as SSRi and EnSRi), respectively, to characterize the role of OsPT8 in P redistribution of rice. In comparison to wild type (WT) and EnSRi lines, SSRi lines under P deficient condition accumulated more P in old blades and less P in young blades, corresponding to attenuated and enriched transcripts of P-responsive genes in old and young blades, respectively. The ratio of total P in young blades to that in old blades decreased from 2.6 for WT to 0.9-1.2 for SSRi lines. During the grain-filling stage, relative to WT, SSRi lines showed the substantial decrease of total P content in both endosperm and embryo, while EnSRi lines showed 40-50% decrease of total P content in embryo but similar P content in endosperm. Taken together, our results demonstrate that OsPT8 plays a critical role in redistribution of P from source to sink organs and P homeostasis in seeds of rice.

Identification of microRNAs in six solanaceous plants and their potential link with phosphate and mycorrhizal signaling

To date, only a limited number of solanaceous miRNAs have been deposited in the miRNA database. Here, genome-wide bioinformatic identification of miRNAs was performed in six solanaceous plants (potato, tomato, tobacco, eggplant, pepper, and petunia). A total of 2,239 miRNAs were identified following a range of criteria, of which 982 were from potato, 496 from tomato, 655 from tobacco, 46 from eggplant, 45 were from pepper, and 15 from petunia. The sizes of miRNA families and miRNA precursor length differ in all the species. Accordingly, 620 targets were predicted, which could be functionally classified as transcription factors, metabolic enzymes, RNA and protein processing proteins, and other proteins for plant growth and development. We also showed evidence for miRNA clusters and sense and antisense miRNAs. Additionally, five Pi starvation- and one arbuscular mycorrhiza (AM)-related cis-elements were found widely distributed in the putative promoter regions of the miRNA genes. Selected miRNAs were classified into three groups based on the presence or absence of P1BS and MYCS cis-elements, and their expression in response to Pi starvation and AM symbiosis was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These results show that conserved miRNAs exist in solanaceous species and they might play pivotal roles in plant growth, development, and stress responses.

How does phosphate status influence the development of the arbuscular mycorrhizal symbiosis

Most terrestrial plant roots form mutualistic symbiosis with soil-borne arbuscular mycorrhizal fungi (AMF), a characteristic feature of which is nutrient exchange between the two symbiotic partners. Phosphate (Pi) is the main benefit the host plants acquired from the AMF. It has long been a common realization that high Pi supply could suppress the AMF development. However, the direct molecular regulatory mechanisms underlying this plant directed suppression are lacking. Here, we reviewed the recent work providing the evidences that high Pi supply induces transcriptional alteration, leading to the inhibition of AMF development at different stages of AM symbiosis, and gave our view on potential cross-talk among Pi starvation, AM as well as phytohormone signaling.