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Interdependent metabolic and transport processes of carbon (C) and nitrogen (N) regulate plant growth and development, while the regulatory pathways remain poorly defined. We previously reported that rice circadian clock N-mediated heading date-1 (Nhd1) regulates growth duration-dependent N use efficiency. Here, we report that knockout of Nhd1 in rice reduced the rate of photosynthesis and the sucrose ratio of sheaths to blades, but increased the total C to N ratio and free amino acids. Leaf RNA-seq analysis indicated that mutation of Nhd1 dramatically altered expression of the genes linked to starch and sucrose metabolism, circadian rhythm, and amino acid metabolic pathways. We identified that Nhd1 can directly activate the transcriptional expression of sucrose transporter-1 (OsSUT1). Knockout of Nhd1 suppressed OsSUT1 expression, and both nhd1 and ossut1 mutants showed similar shorter height, and lower shoot biomass and sucrose concentration in comparison with the wild type, while overexpression of OsSUT1 can restore the defective sucrose transport and partially ameliorate the reduced growth of nhd1 mutants. The Nhd1-binding site of the OsSUT1 promoter is conserved in all known rice genomes. The positively related variation of Nhd1 and OsSUT1 expression among randomly selected indica and japonica varieties suggests a common regulatory module of Nhd1–OsSUT1-mediated C and N balance in rice.

Plants have evolved delicate systems for stimulating or inhibiting inorganic phosphate (Pi) uptake in response to the fluctuating Pi availability in soil. However, the negative regulators inhibiting Pi uptake at the transcriptional level are largely unexplored. Here, we functionally characterized a transcription factor in rice (Oryza sativa), OsWRKY10. OsWRKY10 encodes a nucleus-localized protein and showed preferential tissue localization. Knockout of OsWRKY10 led to increased Pi uptake and accumulation under Pi-replete conditions. In accordance with this phenotype, OsWRKY10 was transcriptionally induced by Pi, and a subset of PHOSPHATE TRANSPORTER 1 (PHT1) genes were up-regulated upon its mutation, suggesting that OsWRKY10 is a transcriptional repressor of Pi uptake. Moreover, rice plants expressing the OsWRKY10–VP16 fusion protein (a dominant transcriptional activator) accumulated even more Pi than oswrky10. Several lines of biochemical evidence demonstrated that OsWRKY10 directly suppressed OsPHT1;2 expression. Genetic analysis showed that OsPHT1;2 was responsible for the increased Pi accumulation in oswrky10. Furthermore, during Pi starvation, OsWRKY10 protein was degraded through the 26S proteasome. Altogether, the OsWRKY10–OsPHT1;2 module represents a crucial loop in the Pi signaling network in rice, inhibiting Pi uptake when there is ample Pi in the environment.

High-affinity potassium (K+) transporters HAK/KUP/KT have been identified in all genome-sequenced terrestrial plants. They play important role in K+acquisition, translocation and enhancing salt tolerance. Here we report that the plasma-membrane-located OsHAK18 functions in K+and sodium (Na+) circulation and sugar translocation in rice. OsHAK18 is expressed mainly though not exclusively in vascular tissues and in the phloem in particular. Knockout (KO) ofOsHAK18reduced K+concentration in phloem sap and in the root but increased K+accumulation in the shoot of both Nipponbare and Zhonghua11 cultivars, while overexpression (OX) ofOsHAK18driven by its endogenous promoter increased K+concentration in phloem sap and in roots, and promoted Na+retrieval from shoot to root under salt stress. Split-root experimental analysis for rubidium (Rb+) uptake and circulation indicated thatOsHAK18-OX promoted Rb+translocation from shoot to root. In addition,OsHAK18-KO increased whileOsHAK18-OX reduced soluble sugar content in shoot while exerting opposite effects on the sugar concentration in the phloem and its content in the root. Moreover,OsHAK18-OX increased dramatically grain yield and K+physiological utilization efficiency. Taken together, our results suggest that – unlike other OsHAKs analyzed heretofore – OsHAK18 is critical to K+and Na+circulation from shoot to root and enhances the source-to-sink translocation of photo-assimilates.One sentence summaryRice potassium transporter OsHAK18 mediates shoot-to-root circulation of potassium and sodium and source-to-sink translocation of soluble sugar for improving potassium use efficiency and grain yield.

Interdependent metabolic and transport processes of carbon (C) and nitrogen (N) regulate plant growth and development, while the regulatory pathways remain poorly defined. We previously reported that a rice circadian clock N-mediated heading date-1 (Nhd1) regulates growth-duration-dependent N use efficiency. Here, we report that knockout of Nhd1 in rice reduced photosynthesis rate and sucrose ratio of sheaths to blades but increased total C to N ratio and free amino acids. Leaf RNA-seq analysis indicated that mutation of Nhd1 dramatically altered expression of the genes linking to starch and sucrose metabolism, circadian rhythm, and amino acid metabolic pathways. We identified that Nhd1 can directly activate the transcriptional expression of sucrose transporter-1 (OsSUT1). Knockout of Nhd1 suppressed OsSUT1 expression and both nhd1 and ossut1 mutants showed similar shorter height, lower shoot biomass and sucrose concentration in comparison to wild type, while overexpression of OsSUT1 can restore the defective sucrose transport and partially ameliorate the reduced growth of nhd1 mutants. Nhd1-binding site of OsSUT1 promoter is conserved in all known rice genomes. The positively related variation of Nhd1 and OsSUT1 expression among randomly selected Indica and Japonica varieties suggests a common regulatory module of Nhd1-OsSUT1 mediated C and N balance in rice.

As sessile organisms, plants rely on their roots for anchorage and uptake of water and nutrients. Plant root is an organ showing extensive morphological and metabolic plasticity in response to diverse environmental stimuli including nitrogen (N) and phosphorus (P) nutrition/stresses. N and P are two essential macronutrients serving as not only cell structural components but also local and systemic signals triggering root acclimatory responses. Here, we mainly focused on the current advances on root responses to N and P nutrition/stresses regarding transporters as well as long-distance mobile proteins and peptides, which largely represent local and systemic regulators, respectively. Moreover, we exemplified some of the potential pitfalls in experimental design, which has been routinely adopted for decades. These commonly accepted methods may help researchers gain fundamental mechanistic insights into plant intrinsic responses, yet the output might lack strong relevance to the real situation in the context of natural and agricultural ecosystems. On this basis, we further discuss the established-and yet to be validated-improvements in experimental design, aiming at interpreting the data obtained under laboratory conditions in a more practical view.

Plant acclimatory responses to phosphate (Pi) starvation stress include the accumulation of carbohydrates, namely sugar and starch. However, whether altered endogenous carbohydrate profile could in turn affect plant Pi starvation responses remains widely unexplored. Here, two genes encoding the large and small subunits of an ADP-glucose pyrophosphorylase (AGP) in rice (Oryza sativa), AGP Large Subunit 1 (AGPL1) and AGP Small Subunit 1 (AGPS1), were functionally characterized with regard to maintenance of phosphorus (P) homeostasis and regulation of Pi starvation signaling. AGPL1 and AGPS1 were both positively responsive to nitrogen (N) or Pi deprivation, and expressed in almost all the tissues except in the meristem and mature zones of root. AGPL1 and AGPS1 physically interacted in chloroplast, and catalyzed the rate-limiting step of starch biosynthesis. Low-N- (LN) and low-Pi (LP)-triggered starch accumulation in leaves was impaired in agpl1, agps1 and apgl1 agps1 mutants compared with the wild-type plants. By contrast, mutation of AGPL1 and/or AGPS1 led to an increase in the content of the major sugar, sucrose, in leaf sheath and root under control and LN conditions. Moreover, the Pi accumulation was enhanced in the mutants under control and LN conditions, but not LP conditions. Notably, the LN-induced suppression of Pi accumulation was compromised attributed to the mutation of AGPL1 and/or AGPS1. Furthermore, the increased Pi accumulation was accompanied by the specific suppression of OsSPX2 and activation of several Pi transporter genes. These results indicate that a balanced level of carbohydrates is vital for maintaining plant P homeostasis.

Plants adjust root architecture and nitrogen (N) transporter activity to meet the variable N demand, but their integrated regulatory mechanism remains unclear. We have previously reported that a floral factor in rice (Oryza sativa), N-mediated heading date-1 (Nhd1), regulates flowering time. Here, we show that Nhd1 can directly activate the transcription of the high-affinity ammonium ([Formula: see text]) transporter 1;3 (OsAMT1;3) and the dual affinity nitrate ([Formula: see text]) transporter 2.4 (OsNRT2.4). Knockout of Nhd1 inhibited root growth in the presence of [Formula: see text] or a low concentration of [Formula: see text]. Compared to the wild-type (WT), nhd1 and osamt1;3 mutants showed a similar decrease in root growth and N uptake under low [Formula: see text] supply, while nhd1 and osnrt2.4 mutants showed comparable root inhibition and altered [Formula: see text] translocation in shoots. The defects of nhd1 mutants in [Formula: see text] uptake and root growth response to various N supplies were restored by overexpression of OsAMT1;3 or OsNRT2.4. However, when grown in a paddy field with low N availability, nhd1 mutants accumulated more N and achieved a higher N uptake efficiency (NUpE) due to the delayed flowering time and prolonged growth period. Our findings reveal a molecular mechanism underlying the growth duration-dependent NUpE.

Summary Plant Phosphate Transporter 1 (PHT1) proteins, probably the only influx transporters for phosphate (Pi) uptake, are partially degraded on sufficient Pi levels to prevent excessive Pi accumulation. Therefore, the basal/constitutive expression level of PHT1 genes is vital for maintaining Pi uptake under Pi‐replete conditions.Rice (Oryza sativa) OsPHT1;1 is a unique gene as it is highly expressed and not responsive to Pi, however the mechanism for maintaining its basal/constitutive expression remains unknown. Using biochemical and genetic approaches, we identified and functionally characterised the transcription factors maintaining the basal/constitutive expression of OsPHT1;1.OsWRKY21 and OsWRKY108 interact within the nucleus and both bind to the W‐box in the OsPHT1;1 promoter. Overexpression of OsWRKY21 or OsWRKY108 led to increased Pi accumulation, resulting from elevated expression of OsPHT1;1. By contrast, oswrky21 oswrky108 double mutants showed decreased Pi accumulation and OsPHT1;1 expression in a Pi‐dependent manner. Moreover, similar to ospht1;1 mutants, plants expressing the OsWRKY21–SRDX fusion protein (a chimeric dominant suppressor) were impaired in Pi accumulation in Pi‐replete roots, accompanied by downregulation of OsPHT1;1 expression.Our findings demonstrated that rice WRKY transcription factors function redundantly to promote Pi uptake by activating OsPHT1;1 expression under Pi‐replete conditions, and represent a novel pathway independent of the central Pi signalling system.

Inorganic phosphate (Pi) is the predominant form of phosphorus (P) readily accessible to plants, and Pi Transporter 1 (PHT1) genes are the major contributors to root Pi uptake. However, the mechanisms underlying the transport and recycling of Pi within plants, which are vital for optimizing P use efficiency, remain elusive. Here, we characterized a functionally unknown rice (Oryza sativa) PHT1 member barely expressed in roots, OsPHT1;7. Yeast complementation and Xenopus laevis oocyte assay demonstrated that OsPHT1;7 could mediate Pi transport. Reverse-transcription quantitative polymerase chain reaction and histochemical analyses showed that OsPHT1;7 was preferentially expressed in source leaves and nodes. A further fine-localization analysis by immunostaining showed that OsPHT1;7 expression was restricted in the vascular bundle (VB) sheath and phloem of source leaves as well as in the phloem of regular/diffuse- and enlarged-VBs of nodes. In accordance with this expression pattern, mutation of OsPHT1;7 led to increased and decreased P distribution in source (old leaves) and sink organs (new leaves/panicles), respectively, indicating that OsPHT1;7 is involved in P redistribution. Furthermore, OsPHT1;7 showed an overwhelmingly higher transcript abundance in anthers than other PHT1 members, and ospht1;7 mutants were impaired in P accumulation in anthers but not in pistils or husks. Moreover, the germination of pollen grains was significantly inhibited upon OsPHT1;7 mutation, leading to a >80% decrease in seed-setting rate and grain yield. Taken together, our results provide evidence that OsPHT1;7 is a crucial Pi transporter for Pi transport and recycling within rice plants, stimulating both vegetative and reproductive growth.

Grains are the major sink of phosphorus (P) in cereal crops, accounting for 60-85% of total plant P, but the mechanisms underlying P loading into the grains are poorly understood. We functionally characterized a transporter gene required for the distribution of P to the grains in barley (Hordeum vulgare), HvSPDT (SULTR-like phosphorus distribution transporter). HvSPDT encoded a plasma membrane-localized Pi/H

Phosphorus (P), which is taken up by plants as inorganic phosphate (Pi), is one of the most important macronutrients for plant growth and development. Meanwhile, it determines plant architecture in several ways, including leaf inclination. However, the molecular basis underlying the crosstalk between the signaling pathways of plant P homeostasis and architecture maintenance remains elusive. We recently characterized a WRKY transcription factor, OsWRKY108, in rice (Oryza sativa). It functions redundantly with OsWRKY21 to promote Pi uptake in response to Pi supply. Overexpression of either OsWRKY108 or OsWRKY21 led to up-regulation of Pi transporter genes and thus enhanced Pi accumulation. By contrast, transgenic rice plants expressing OsWRKY21-SRDX (a fusion protein transforming OsWRKY21 from an activator into a dominant repressor) but not the OsWRKY108-SRDX fusion showed decreased Pi accumulation under Pi-replete conditions. Here, we report that OsWRKY108 acts as a positive regulator of leaf inclination. OsWRKY108 overexpressors showed increased leaf inclination and OsWRKY108-SRDX plants showed an erect-leaf phenotype, irrespective of the Pi regimes. Nevertheless, the response of leaf inclination to Pi starvation was largely impaired upon OsWRKY108 overexpression. Moreover, in both OsWRKY108-SRDX plants and OsWRKY108 overexpressors, the ‘percentage of leaf angle alteration relative to wild-type’ under Pi-starvation condition was more significant than that under Pi-replete condition. These results suggest that the regulation of OsWRKY108 on leaf inclination is in part dependent on Pi availability. Altogether, our findings demonstrate that OsWRKY108 is an integrative regulator of P homeostasis and leaf inclination, serving as a link between plant nutrient signaling and developmental cues.

Plant roots rely on inorganic orthophosphate (Pi) transporters to acquire soluble Pi from soil solutions that exists at micromolar levels in natural ecosystems. Here, we functionally characterized a rice (Oryza sativa) Pi transporter, Os Phosphate Transporter-1;3 (OsPHT1;3), that mediates Pi uptake, translocation, and remobilization. OsPHT1;3 was directly regulated by Os Phosphate Starvation Response-2 and, in response to Pi starvation, showed enhanced expression in young leaf blades and shoot basal regions and even more so in roots and old leaf blades. OsPHT1;3 was able to complement a yeast mutant strain defective in five Pi transporters and mediate Pi influx in Xenopus laevis oocytes. Overexpression of OsPHT1;3 led to increased Pi concentration both in roots and shoots. However, unlike that reported for other known OsPHT1 members that facilitate Pi uptake at relatively higher Pi levels, mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only when external Pi concentration was below 5 μm. Moreover, in basal nodes, the expression of OsPHT1;3 was restricted to the phloem of regular vascular bundles and enlarged vascular bundles. An isotope labeling experiment with (32)P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from source to sink leaves. Furthermore, overexpression and mutation of OsPHT1;3 led to reciprocal alteration in the expression of OsPHT1;2 and several other OsPHT1 genes. Yeast-two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays all demonstrated a physical interaction between OsPHT1;3 and OsPHT1;2. Taken together, our results indicate that OsPHT1;3 acts as a crucial factor for Pi acquisition, root-to-shoot Pi translocation, and redistribution of phosphorus in plants growing in environments with extremely low Pi levels.

Symbiotic nitrogen (N2 ) fixation plays a vital role in sustainable agriculture. Efficient N2 fixation requires various materials, including phosphate (Pi); however, the molecular mechanism underlying the transport of Pi into nodules and bacteroids remains largely unknown. A nodule-localized Pi transporter, GmPT7, was functionally characterized in soybean (Glycine max) and its role in N2 fixation and yield was investigated via composite and whole transgenic plants. GmPT7 protein was localized to the plasma membrane and showed transport activity for Pi in yeast. Altered expression of GmPT7 changed 33 Pi uptake from rhizosphere and translocation to bacteroids. GmPT7 was mainly localized to the outer cortex and fixation zones of the nodules. Overexpression of GmPT7 promoted nodulation, and increased plant biomass, shoot nitrogen and phosphorus content, resulting in improved soybean yield by up to 36%. Double suppression of GmPT5 and GmPT7 led to nearly complete elimination of nodulation and over 50% reduction in plant biomass, shoot nitrogen and phosphorus content, indicating that both GmPT7 and GmPT5 contribute to Pi transport for N2 fixation. Taken together, our results indicate that GmPT7 is a transporter responsible for direct Pi entry to nodules and further to fixation zones, which is required for enhancing symbiotic N2 fixation and grain yield of soybean.

OsMYB1 affects phosphate homeostasis and root development by independent regulation of genes involved in phosphate starvation signaling and gibberellic acid biosynthesis., The adaptive responses of plants to phosphate (Pi) starvation stress are fine-tuned by an elaborate regulatory network. In this study, we identified and characterized a novel Pi starvation-responsive gene, MYB1, encoding an R2R3-type transcription factor in rice. MYB1 was transcriptionally induced in leaf sheaths and old leaf blades. It was localized to the nucleus and expressed mainly in vascular tissues. Mutation of MYB1 led to an increase in Pi uptake and accumulation, accompanied by altered expression of a subset of Pi transporters and several genes involved in Pi starvation signaling. Furthermore, MYB1 affected the elongation of the primary root in a Pi-dependent manner and lateral roots in a Pi-independent manner. Moreover, gibberellic acid (GA)-triggered lateral root elongation was largely suppressed in wild-type plants under Pi starvation conditions, whereas this suppression was partially rescued in myb1 mutant lines, correlating with the up-regulation of a GA biosynthetic gene upon MYB1 mutation. Taken together, the findings of this study highlight the role of MYB1 as a regulator involved in both Pi starvation signaling and GA biosynthesis. Such a co-regulator might have broad implications for the study of cross-talk between nutrient stress and other signaling pathways.

Symbiotic nitrogen (N

Our previous studies demonstrated that chloroplastic CO2 concentration (Cc) is not sufficient under high-nitrogen (N) supply in rice plants. In this research, we studied how aquaporins- (AQPs) mediated Cc under different N-supply levels. A hydroponic experiment was conducted in a greenhouse with three different N levels (low N, 0.71 mM; intermediate N, 2.86 mM; and high N, 7.14 mM) in a rice cultivar (Oryza sativa cv. Shanyou 63) and with an ospip1;1 mutant (Oryza sativa cv. Nipponbare). The photosynthetic nitrogen-use efficiency (PNUE) decreased with increasing leaf-N content. Under high-N supply, the estimated Cc was significantly lower than the theoretical Cc and the specific Rubisco activity (carboxylation efficiency/Rubisco content, CE/Rubisco) decreased, because of a decrease of relative CO2 diffusion conductance (total CO2 diffusion conductance/leaf-N content, gt /N) in mesophyll cells. Real Time Quantitative PCR (Q-RT-PCR) showed that most OsPIP1s and OsPIP2s expression were downregulated under the high-N supply. Furthermore, Cc and gm decreased in the ospip1;1 mutant line compared with that of the wild-type plant. It was concluded that under high-N supply, the decreased PNUE was associated with non-sufficient Cc, mediated by AQP in mesophyll conductance.

Many terrestrial plants can form root symbiosis with beneficial microorganisms for enhancing uptake of mineral nutrients or increasing fitness to adverse environmental challenges. Arbuscular mycorrhizal (AM) symbiosis that is formed by AM fungi and the roots of vascular flowering plants is the most widespread mutualistic associations in nature. As a typical endosymbiosis, AM interactions involves the differentiation of both symbionts to create novel symbiotic interfaces within the root cells, and requires a continuous nutrient exchange between the two partners. AM plants have two pathways for nutrient uptake, either direct uptake via the root hairs and root epidermis at the plant-soil interface, or indirectly through the AM fungal hyphae at the plant-fungus interface. Over the last few years, great progress has been made in deciphering the mechanisms underlying the AM-mediated modulation of nutrient uptake processes, and an increasing number of plant and fungal genes responsible for transporting nutrients from the soil or across the intraradical symbiotic interfaces have been identified and functionally characterized. Here, we summarize the recent advances in the nitrogen uptake, assimilation and translocation in the AM symbiosis, and also explore the current understanding of how the N status and interplay with C and P in modulating the development of AM associations.

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.

Auxin is well known to be a key regulator that acts in almost all physiological processes during plant growth, and in interactions between plants and microbes. However, to date, the regulatory mechanisms underlying auxin-mediated plant-arbuscular mycorrhizal (AM) fungi symbiosis have not been well deciphered. Previously we identified a GH3 gene, SlGH3.4, strongly responsive to both auxin induction and mycorrhizal symbiosis. Here, we reported a refined dissection of the SlGH3.4 promoter activity using the β-glucuronidase (GUS) reporter. The SlGH3.4 promoter could drive GUS expression strongly in mycorrhizal roots of soybean and rice plants, and in IAA-treated soybean roots, but not in IAA-treated rice roots. A promoter deletion assay revealed three cis-acting motifs, i.e. the auxin-responsive element, AuxRE, and two newly identified motifs named MYCRS1 and MYCRS2, involved in the activation of auxin- and AM-mediated expression of SlGH3.4. Deletion of the AuxRE from the SlGH3.4 promoter caused almost complete abolition of GUS staining in response to external IAA induction. Seven repeats of AuxRE fused to the Cauliflower mosaic virus (CaMV) 35S minimal promoter could direct GUS expression in both IAA-treated and AM fungal-colonized roots of tobacco plants. Four repeats of MYCRS1 or MYCRS2 fused to the CaMV35S minimal promoter was sufficient to drive GUS expression in arbuscule-containing cells, but not in IAA-treated tobacco roots. In summary, our results offer new insights into the molecular mechanisms underlying the potential cross-talk between the auxin and the AM regulatory pathways in modulating the expression of AM-responsive GH3 genes in diverse mycorrhizal plants.

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.

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.

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.

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.

The plasma membrane (PM) H+ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H+ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H+ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (Km) of PM H+ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H+ ATPase in P deficient roots was related to a slightly increase of PM H+ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H+-ATPase and H+ pump, which contributed to the rhizosphere acidification in rice roots.

• 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.

Allelochemicals from root exudates or decaying residues of watermelon plant may be related to watermelon fusarium wilt. The aim of this work was to study the effect of an artificially applied allelochemical, ferulic acid, on in vitro Fusarium oxysporum f. sp. niveum (FON), a causal pathogen of plant wilting in the laboratory setting. The results showed that ferulic acid inhibited growth at high concentrations. The biomass was reduced by 71.6% and the conidial germinate rate was decreased by 100%, while mycotoxin production by FON was increased by 227.7% at the highest concentration (1600 mg L−1). Activities of hydrolytic enzymes related to pathogenicity were also affected. It is concluded that ferulic acid at commonly found concentrations inside plants suppressed the growth of pathogen F. oxysporum f. sp. niveum.

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.

It has been almost 25 years since the first report of the gene encoding a high-affinity phosphate transporter (PT), PHO84, in yeast. Since then, an increasing number of yeast PHO84 homologs as well as other genes encoding proteins with phosphate (Pi) transport activities have been identified and functionally characterized in diverse plant species. Great progress has been made also in deciphering the molecular mechanism underlying the regulation of the abundance and/or activity of these genes and their products. The regulatory genes affect plant Pi homeostasis commonly through direct or indirect regulation of the abundance of PTs at different levels. However, little has been achieved in the use of PTs for developing genetically modified crops with high phosphorus use efficiency (PUE). This might be a consequence of overemphasizing Pi uptake from the rhizosphere and lack of knowledge about the roles of PTs in Pi transport and recycling within the plant that are required to optimize PUE. Here, we mainly focused on the genes encoding proteins with Pi transport activities and the emerging understanding of their regulation at the transcriptional, post-transcriptional, translational, and post-translational levels. In addition, we propose potential strategies for effective use of PTs in improving plant growth and development.

Our previous studies demonstrated that chloroplastic CO

Plant phosphorus (P) diagnosis is widely used for monitoring P status and guiding P fertilizer application in field conditions. The common methods for predicting plant response to P are time- and labour-consuming chemical measurements of the extractable soil P and plant P concentrations. In this study, we successfully generated a visual reporter system in tobacco (Nicotiana tabacum L.) to monitor plant P status by expressing of a Purple gene (Pr) isolated from cauliflower (Brassica oleracea var botrytis) driven by the promoter (Pro) of OsPT6, a P-starvation-induced rice gene. The leaves of OsPT6pro::Pr (PT6pro::Pr) transgenic tobacco continuously turned into dark purple with the increase of duration and severity of P deficiency, and recovered rapidly to basal green colour upon resupply of P. The expression of several anthocyanin biosynthesis involving genes was strongly activated in the transgenic tobacco in comparison to wild type under P-deficient condition. Such additive purple colour was not detected by deficiencies of other major- and micronutrients or stresses of salt, drought and cold. There was an extremely high correlation between P concentration and anthocyanin accumulation in the transgenic tobacco leaves. Using a hyperspectral sensing technology, P concentration in the leaves of transgenic plants could be predicted by the reflectance spectra at 554 nm wavelength with approximately 0.16 as the threshold value of the P deficiency. Taken together, the colour-based visual reporter system could be specifically and readily used for monitoring the plant P status by naked eyes and accurately assessed by spectral reflectance.

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.

In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants.