Shubin Sun

Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation.

Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice (Oryza sativa) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant.

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.

Facilitated legume nodulation, phosphate uptake and nitrogen transfer by arbuscular inoculation in an upland rice and mung bean intercropping system

Intercropping of upland rice with short-duration grain legumes has major advantages in increasing crop yields and soil productivity. However, the contribution of arbuscular mycorrhizas, the common mutualistic symbiosis between most crops and mycorrhizal fungi, is not fully understood in intercropping systems. We assayed the contribution of inoculation of the arbuscular mycorrhizal fungus (AMF) Glomus caledonium on nutrient acquisition and biomass yield. Using the method of plastic film and nylon net partition and tracing 15N transferred between the intercropped upland rice (Oryza sativa ssp. Japonica Nipponbare) and mung bean (Vigna radiata L. Chuanyuan), we compared the intercropping, with separation of the whole root systems by a plastic film, with and without a barrier of nylon net to allow penetration of the fungal hyphae. Intercropping significantly improved the formation of arbuscular mycorrhizas, particularly in the upland rice roots. The improved formation of mycorrhizas by the intercropping increased total P uptake by 57% in rice, total P and N acquisition by 65% and 64% respectively in mung bean, and nodulation by 54% in mung bean. The percentage of total 15N transfer from mung bean to rice leaves was increased from 5.4% to 15.7% by inoculation with AMF. In contrast, there was only 2.7% of 15N transfer from rice to mung bean and no AMF effect on N transfer. It is concluded that cereal and legume crop intercropping increase mycorrhiza formation, which in turn improves nodulation, N and P acquisition and N transfer in the legumes.

Physiological and Molecular Responses of Nitrogen-starved Rice Plants to Re-supply of Different Nitrogen Sources

Rice grown under anaerobic waterlogged soil condition takes up ammonium as a major source of nitrogen (N). A substantial quantity of nitrate is taken up by rice when aeration conditions prevail. This work compared the physiological and molecular responses of N-depleted rice (Oryza sativa L. japonica ssp. cv. Wuyunjing) to re-supply of different N sources in a hydroponic system. Expression of six genes known to be related to N uptake and assimilation in roots was analyzed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). N starvation increased root growth but decreased shoot biomass and nitrate reductase (NR) activity. In comparison to nitrate as N source, ammonium tended to inhibit root growth but significantly improved shoot growth, total N uptake, and, particularly, the translocation of N to the above ground parts. Replacing half of the ammonium by nitrate in the single ammonium solution increased glutamine synthetase (GS) activity and total N uptake. This replacement enhanced expression of OsAMT1;3 but decreased the expression of OsAMT1;2 in the roots. Likewise, replacing half of the nitrate by ammonium in the single nitrate solution, increased NR activity and expression of an NR gene (OsNR1) but suppressed expression of OsNRT2 in the roots. Expression of both OsAMT1;1 and a GS gene (OsGS1;1) was relatively constant and not affected by the N form or starvation in the rice roots. Predominant beneficial effects of dual supply of the two forms of N in comparison with a single form of N either as ammonium or as nitrate might be due to improvement of ammonium uptake due to nitrate presence near the roots and enhanced reduction of nitrate in leaves by ammonium.

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.

Involvement of OsPht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice

Phosphate (Pi) transporters mediate acquisition and transportation of Pi within plants. Here, we investigated the functions of OsPht1;4 (OsPT4), one of the 13 members of the Pht1 family in rice. Quantitative real-time RT-PCR analysis revealed strong expression of OsPT4 in roots and embryos, and OsPT4 promoter analysis using reporter genes confirmed these findings. Analysis using rice protoplasts showed that OsPT4 localized to the plasma membrane. OsPT4 complemented a yeast mutant defective in Pi uptake, and also facilitated increased accumulation of Pi in Xenopus oocytes. Further, OsPT4 genetically modified (GM) rice lines were generated by knockout/knockdown or over-expression of OsPT4. Pi concentrations in roots and shoots were significantly lower and higher in knockout/knockdown and over-expressing plants, respectively, compared to wild-type under various Pi regimes. (33) Pi uptake translocation assays corroborated the altered acquisition and mobilization of Pi in OsPT4 GM plants. We also observed effects of altered expression levels of OsPT4 in GM plants on the concentration of Pi, the size of the embryo, and several attributes related to seed development. Overall, our results suggest that OsPT4 encodes a plasma membrane-localized Pi transporter that facilitates acquisition and mobilization of Pi, and also plays an important role in development of the embryo in rice.

Effect of Nitrate on Activities and Transcript Levels of Nitrate Reductase and Glutamine Synthetase in Rice

Abstract Real-time polymerase chain reaction analysis was used to compare the effect of NO−3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS), and the transcript levels of two NR genes, OsNia1 and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice (Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ). Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+4 nutrition. Supply of NO−3 increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1. NO−3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO−3 had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO−3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ, indicating a stronger response of NG to NO−3 nutrition than YJ.

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.

OsSIZ1, a SUMO E3 Ligase Gene, is Involved in the Regulation of the Responses to Phosphate and Nitrogen in Rice

SIZ1-mediated SUMOylation regulates hormone signaling as well as abiotic and biotic stress responses in plants. Here, we investigated the expression profile of OsSIZ1 in rice using quantitative reverse transcription-PCR (qRT-PCR) and pOsSIZ1-GUS transgenic plants, and the function of OsSIZ1 in the responses to phosphate and nitrogen using a reverse genetics approach. OsSIZ1 is constitutively expressed throughout the vegetative and reproductive growth of rice, with stronger promoter activities in vascular bundles of culms. ossiz1 mutants had shorter primary roots and adventitious roots than wild-type plants, suggesting that OsSIZ1 is associated with the regulation of root system architecture. Total phosphorus (P) and phosphate (Pi) concentrations in both roots and shoots of ossiz1 mutants were significantly increased irrespective of Pi supply conditions compared with the wild type. Pi concentration in the xylem sap of ossiz1 mutants was significantly higher than that of the wild type under a Pi-sufficient growth regime. Total nitrogen (N) concentrations in the most detected tissues of ossiz1 mutants were significantly increased compared with the wild type. Analysis of mineral contents in ossiz1 mutants indicated that OsSIZ1 functions specifically in Pi and N responses, not those of other nutrients examined, in rice. Further, qRT-PCR analyses revealed that the expression of multiple genes involved in Pi starvation signaling and N transport and assimilation were altered in ossiz1 mutants. Together, these results suggested that OsSIZ1 may act as a regulator of the Pi (N)-dependent responses in rice.