Pi-sufficient Conditions Research Articles

Non-specific Phospholipase C4 Improves Phosphorus Remobilization From Old to Young Leaves in Camelina.

Camelina sativa is regarded as a low-input oilseed crop for versatile food, biofuels and industrial applications with potential production on marginal lands, whereas phosphate (Pi) deficiency greatly reduces camelina seed production. To improve camelina resilience to low P conditions, here we overexpressed the Pi deficiency-induced non-specific phospholipase C4 (NPC4) to test its effect on camelina seed production under different levels of Pi availability. NPC4-overexpressing (OE) plants displayed increased seed yield and oil production, with a greater magnitude of increases under Pi-deficient than Pi-sufficient conditions. NPC4-OE camelina had a higher level of total P and free Pi in young leaves but a lower level in old leaves than in wild-type plants. More Pi was moved from old leaves to young leaves in NPC4-OE than in wild-type plants. NPC4-OE increased the expression of Pi transporter genes, and the increase was greater in old leaves and under Pi-deficient conditions. These data indicate that NPC4 improves camelina growth by promoting Pi remobilization from old to young tissues, revealing a mechanism by which NPC4 mediates plant response to Pi deficiency.

Transcriptome analysis with different leaf blades identifies the phloem-specific phosphate transporter OsPHO1;3 required for phosphate homeostasis in rice.

Phosphate (Pi) is essential for plant growth and development. One strategy to improve Pi use efficiency is to enhance Pi remobilization among leaves. Using transcriptome analysis with first (top) and fourth (down) leaf blades from rice (Oryza sativa) in Pi-sufficient and deficient conditions, we identified 1384 genes differentially expressed among these leaf blades. These genes were involved in physiological processes, metabolism, transport, and photosynthesis. Moreover, we identified the Pi efflux transporter gene, OsPHO1;3, responding to Pi-supplied conditions among these leaf blades. OsPHO1;3 is highly expressed in companion cells of phloem, but not xylem, in leaf blades and induced by Pi starvation. Mutation of OsPHO1;3 led to Pi accumulation in second to fourth leaves under Pi-sufficient conditions, but enhanced Pi levels in first leaves under Pi-deficient conditions. These Pi accumulations in leaves of Ospho1;3 mutants resulted from induction of OsPHT1;2 and OsPHT1;8 in root and reduction of Pi remobilization in leaf blades, revealed by the decreased Pi in phloem of leaves. Importantly, lack of OsPHO1;3 caused growth defects under a range of Pi-supplied conditions. These results demonstrate that Pi remobilization is essential for Pi homeostasis and plant growth irrespective of Pi-supplied conditions, and OsPHO1;3 plays an essential role in Pi remobilization for normal plant growth.

Protein phosphatase 5 mediates plant growth and phosphate homeostasis in rice

Protein phosphorylation is a reversible post-translational modification, and protein phosphatase is the most important regulator for protein dephosphorylation. Protein phosphatase 5 (PP5), which functions in many cellular processes essential for cell survival and growth in animals, has not been functionally reported in rice. In this study, we functionally identified OsPP5 in rice. OsPP5 is localized in both the nucleus and the cytoplasm which functions as a manganese (Mn2+)-dependent phosphatase. OsPP5 is expressed in most tissues of rice and is positively regulated by Pi supply at transcript and protein levels. Compared with the wild-type plants, OsPP5 overexpression lines showed increased Pi concentration under both Pi sufficient and Pi deficient conditions, while OsPP5 RNA interfering plants showed the opposite phenotype. Expression of phosphate transporters OsPT2, OsPT9, and OsPT10 were increased in OsPP5 overexpression plants. Our findings show that OsPP5 plays an important role in rice Pi homeostasis which could be a potential target for improving Pi absorption under low Pi environment in rice.

A cucumber protein, Phloem Phosphate Stress-Repressed 1, rapidly degrades in response to a phosphate stress condition.

Under depleted external phosphate (Pi), many plant species adapt to this stress by initiating downstream signaling cascades. In plants, the vascular system delivers nutrients and signaling agents to control physiological and developmental processes. Currently, limited information is available regarding the direct role of phloem-borne long-distance signals in plant growth and development under Pi stress conditions. Here, we report on the identification and characterization of a cucumber protein, Cucumis sativus Phloem Phosphate Stress-Repressed 1 (CsPPSR1), whose level in the phloem translocation stream rapidly responds to imposed Pi-limiting conditions. CsPPSR1 degradation is mediated by the 26S proteasome; under Pi-sufficient conditions, CsPPSR1 is stabilized by its phosphorylation within the sieve tube system through the action of CsPPSR1 kinase. Further, we discovered that CsPPSR1 kinase was susceptible to Pi starvation-induced degradation in the sieve tube system. Our findings offer insight into a molecular mechanism underlying the response of phloem-borne proteins to Pi-limited stress conditions.

Comparative biochemical and transcriptomic analysis reveals the phosphate-starving tolerance of two ramie varieties

Ramie (Boehmeria nivea L.) is a highly valued fiber crop. Its yield is often limited by lack of available phosphate (Pi) in the soil, but the underlying molecular mechanisms of ramie's response to Pi deficiency remain largely unknown. To investigate how ramie adapts to low Pi stress, we selected a low Pi-tolerant variety (H-5) and a low Pi-sensitive variety (XYL), and conducted a biochemical and transcriptomic analysis on roots and leaves of both varieties. After subjecting the plants to Pi-deficient and Pi-sufficient conditions for 15 days, we found that H-5 exhibited higher dry weight, longer root systems, and higher levels of Pi, galactolipids, and organic acids when subjected to Pi deprivation, compared to XYL. Transcriptomic analysis further revealed that Pi-responsive genes involved in lipid metabolism, Pi transport, organic acid synthesis, and acid phosphatase activities were more induced in the tolerant variety H-5. Furthermore, weighted gene co-expression network analysis (WGCNA) identified five hub genes, including phosphate transporter, SPX domain-containing protein and sulfoquinovosyl transferase, which played key roles in low Pi tolerance in ramie. The present study will broaden our comprehension of the differences and molecular mechanisms of different ramie cultivars in response to Pi starvation, and lay a foundation for future agronomic improvements in ramie and other fiber crops.

OsIPK2, a rice inositol polyphosphate kinase gene, is involved in phosphate homeostasis and root development.

Phosphorus (P) is one kind of growth-limiting nutrient for plants, which is taken up by root tissue from the environment as inorganic phosphate (Pi). To maintain an appropriate status of cellular Pi, plants have developed sophisticated strategies to sense Pi level and modulate their root system architecture (RSA) under the ever-changing growth condition. However, the molecular basis underlying the mechanism remains elusive. Inositol polyphosphate kinase (IPK2) is a key enzyme in the inositol phosphate metabolism pathway, which catalyzes the phosphorylation of IP3 into IP5 by consuming ATP. In this study, the function of a rice inositol polyphosphate kinase gene (OsIPK2) in plant Pi homeostasis and thus physiological response to Pi signal were characterized. As a biosynthetic gene for phytic acid in rice, overexpression of OsIPK2 led to distinct changes of inositol polyphosphate profiles and an excessive accumulation of Pi level in transgenic rice under Pi-sufficient conditions. The inhibitory effects of OsIPK2 on root growth were alleviated by Pi-deficient treatment compared with wild-type plants, suggesting the involvement of OsIPK2 in the Pi-regulated reconstruction of RSA. In OsIPK2-overexpressing plants, the altered acid phosphatase (APases) activities and misregulation of Pi starvation induced (PSI) genes were observed in roots under different Pi supply conditions. Notably, the expression of OsIPK2 also altered the Pi homeostasis and root system architecture in transgenic Arabidopsis. Taken together, our findings demonstrated that OsIPK2 plays an important role in Pi homeostasis and root system architecture adjustment in response to different environmental Pi levels in plant.

Silencing of SlSPX1 and SlSPX2 promote growth and root mycorrhization in tomato (Solanum lycopersicum L.) seedlings

Owing to the essential requirement of phosphorus (P) for growth and development, plants tightly control inorganic phosphate (Pi) homeostasis. SPX-PHR regulatory circuit not only control phosphate homeostasis responses but also root mycorrhization by arbuscular mycorrhiza (AM) fungi. Besides sensing Pi deficiency, SPX (SYG1/Pho81/XPR1) proteins also control the transcription of P starvation inducible (PSI) genes by blocking the activity of PHR1 (PHOSPHATE STARVATION RESPONSE1) homologs in plants under Pi-sufficient conditions. However, the roles of SPX members in Pi homeostasis and AM fungi colonization remain to be fully recognized in tomato. In this study, we identified 17 SPX-domain containing members in the tomato genome. Transcript profiling revealed the high Pi-specific nature of their activation. Four SlSPX members have also induced in AM colonized roots. Interestingly, we found that SlSPX1 and SlSPX2 are induced by P starvation and AM fungi colonization. Further, SlSPX1 and SlSPX2 exhibited varying degrees of interaction with the PHR homologs in this study. Virus-induced gene silencing-based (VIGS) transcript inhibition of these genes alone or together promoted the accumulation of higher total soluble Pi in tomato seedlings and improved their growth. It also enhanced AM fungi colonization in the roots of SlSPX1 and SlSPX2 silenced seedlings. Overall, the present study provides evidence in support of SlSPX members being good candidates for improving AM fungi colonization potential in tomato.

Overexpression of ClWRKY48 from Cunninghamia lanceolata improves Arabidopsis phosphate uptake.

Our findings suggested that ClWRKY48 promoted the expression level of Arabidopsis phosphate transporter genes, enhanced phosphate uptake, and delayed the transition from the vegetative stage to the reproductive phase in Arabidopsis. Phosphorus (P) is an essential mineral for plants that influences their growth and development. ClWRKY48, one of the most highly expressed genes in the leaf, was identified by RT-PCR from Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] (C. lanceolata). Furthermore, when treating C. lanceolata with increasing phosphate (Pi) concentration, the expression level of ClWRKY48 rose in leaves, the trends followed the increasing phosphate concentration treatment. ClWRKY48 is a transcription factor in C. lanceolata, according to the results of a yeast one hybridization experiment. Based on subcellular localization studies, ClWRKY48 is a nuclear-localized protein. Under Pi deficiency conditions, the phosphorus concentration of ClWRKY48 overexpressing Arabidopsis increased by 43.2-51.1% compared to the wild-type.Moreover, under Pi limiting conditions, the phosphate transporter genes AtPHT1;1 (Arabidopsis Phosphate transporter 1;1), AtPHT1;4, and AtPHO1 (Arabidopsis PHOSPHATE 1) were expressed 2.1-2.5, 2.2-2.7, and 6.7-7.3-fold greater than the wild-type in ClWRKY48 transgenic Arabidopsis, respectively. Under Pi-sufficient conditions, the phosphorus concentration and phosphate transporter genes of ClWRKY48 overexpression in Arabidopsis are not significantly different from the wild type. These findings indicated that ClWRKY48 increased phosphate absorption in transgenic Arabidopsis. Furthermore, compared to the wild type, the ClWRKY48 transgenic Arabidopsis not only had a delayed flowering time characteristic but also had lower expression of flowering-related genes AtFT (FLOWERING LOCUS T), AtFUL (FRUITFUL), and AtTSF (TWIN SISTER OF FT). Our findings show that ClWRKY48 enhances phosphate absorption and slows the transition from the vegetative to the reproductive stage in ClWRKY48 transgenic Arabidopsis.

Mutation of OsLPR3 Enhances Tolerance to Phosphate Starvation in Rice.

Low Phosphate Root (LPR) encodes a protein localized to the endoplasmic reticulum (ER) and cell wall. This gene plays a key role in responding to phosphate (Pi) deprivation, especially in remodeling the root system architecture (RSA). An identification and expression analysis of the OsLPR family in rice (Oryza sativa) has been previously reported, and OsLPR5, functioning in Pi uptake and translocation, is required for the normal growth and development of rice. However, the role of OsLPR3, one of the five members of this family in rice, in response to Pi deficiency and/or in the regulation of plant growth and development is unknown. Therefore, in this study, the roles of OsLPR3 in these processes were investigated, and some functions were found to differ between OsLPR3 and OsLPR5. OsLPR3 was found to be induced in the leaf blades, leaf sheaths, and roots under Pi deprivation. OsLPR3 overexpression strongly inhibited the growth and development of the rice but did not affect the Pi homeostasis of the plant. However, oslpr3 mutants improved RSA and Pi utilization, and they exhibited a higher tolerance to low Pi stress in rice. The agronomic traits of the oslpr3 mutants, such as 1000-grain weight and seed length, were stimulated under Pi-sufficient conditions, indicating that OsLPR3 plays roles different from those of OsLPR5 during plant growth and development, as well as in the maintenance of the Pi status of rice.

Diverse functions of GmNLA1 members in controlling phosphorus homeostasis highlight coordinate response of soybean to nitrogen and phosphorus availability

Nitrogen (N) and phosphorus (P) are two essential mineral nutrients for plant growth, which are required in relative high amount in plants. Plants have evolved a series of strategies for coordinately acquiring and utilizing N and P. However, physiological and molecular mechanisms underlying of N and P interactions remain largely unclear in soybean (Glycine max). In this study, interactions of N and P were demonstrated in soybean as reflected by significant increases of phosphate (Pi) concentration in both leaves and roots by N deficiency under Pi sufficient conditions. A total of four nitrogen limitation adaptation (NLA), encoding RING-type E3 ubiquitin ligase were subsequently identified in soybean genome. Among them, transcription of GmNLA1-1 and GmNLA1-3 was decreased in soybean by N starvation under Pi sufficient conditions, not for GmNLA1-2. Suppression of all three GmNLA1 members was able to increase Pi concentration regardless of the P and N availability in the growth medium, but decrease fresh weight under normal conditions in soybean hairy roots. However, comparted to changes in control lines at two N levels, N deficiency only resulted in a relatively higher increase of Pi concentration in GmNLA1-1 or GmNLA1-3 suppression lines, strongly indicating that GmNLA1-1 and GmNLA1-3 might regulate P homeostasis in soybean response to N starvation. Taken together, our result suggest that redundant and diverse functions present in GmNLA1 members for soybean coordinate responses to P and N availability, which mediate P homeostasis.

SbPHO2, a conserved Pi starvation signalling gene, is involved in the regulation of the uptake of multiple nutrients in sorghum

Sorghum is one of the five most productive crops worldwide, but its yield is seriously limited by phosphate (Pi) availability. Although inorganic Pi signalling is well studied in Arabidopsis and rice, it remains largely unknown in sorghum. The sorghum sbpho2 mutant was identified, showing leaf necrosis and short roots. Map-based cloning identified SbPHO2 as Sobic.009G228100, an E2 conjugase gene that is a putative orthologue of the PHO2 genes in rice and Arabidopsis, which play important roles in Pi signalling. Pi starvation experiments and transformation of SbPHO2 into the rice ospho2 mutant further revealed that SbPHO2 is likely involved in Pi accumulation and root architecture alteration in sorghum. qRTPCR results showed that SbPHO2 was expressed in almost the entire plant, especially in the leaves. Furthermore, some typical Pi starvation-induced genes were induced in sbpho2 even under Pi-sufficient conditions, including Pi transporters, SPXs, phosphatases and lipid composition alteration-related genes. In addition to P accumulation in the shoots of sbpho2, concentrations of N, K, and other metal elements were also altered significantly in the sbpho2 plants. Nitrate uptake was also suppressed in the sbpho2 mutant. Consistent with this finding, the expression of several nitrate-, potassium- and other metal element-related genes was also altered in sbpho2. Furthermore, the results indicated that N-dependent control of the P starvation response is regulated via SbPHO2 in sorghum. Our results suggest that SbPHO2 participates in the regulation of the absorption of multiple nutrients, although PHO2 is a crucial and conserved component of Pi starvation signalling.

SHORT-ROOT stabilizes PHOSPHATE1 to regulate phosphate allocation in Arabidopsis.

The coordinated distribution of inorganic phosphate (Pi) between roots and shoots is an important process that plants use to maintain Pi homeostasis. SHORT-ROOT (SHR) is well characterized for its function in root radial patterning. Here we demonstrate a role of SHR in controlling Pi allocation from root to shoot by regulating PHOSPHATE1 in the root differentiation zone. We recovered a weak mutant allele of SHR in Arabidopsis that accumulates much less Pi in the shoot and shows a constitutive Pi starvation response under Pi-sufficient conditions. In addition, Pi starvation suppresses SHR protein accumulation and releases its inhibition on the HD-ZIP III transcription factor PHB. PHB accumulates and directly binds the promoter of PHOSPHATE2 to upregulate its transcription, resulting in PHOSPHATE1 degradation in the xylem-pole pericycle cells. Our findings reveal a previously unrecognized mechanism of how plants regulate Pi translocation from roots to shoots.

PtoWRKY40 interacts with PtoPHR1-LIKE3 while regulating the phosphate starvation response in poplar.

Plants usually suffer from phosphorus starvation because of the low inorganic phosphate (Pi) status of most soils. To cope with this, plants have evolved an adaptive phosphate starvation response (PSR) which involves both developmental and metabolic changes regulated mainly by PHOSPHATE STARVATION RESPONSE1 (PHR1) and its homologs. Here, we elucidated how perennial woody plants, such as poplars (Populus spp.), respond to low-Pi stress. We first performed RNA-seq analysis of low-Pi-treated poplars and identified PtoWRKY40 is rapidly downregulated and protein degraded after stress. Overexpressing and knocking-down PtoWRKY40 downregulated and upregulated the expression of Pi starvation signaling genes, respectively, such as PHOSPHATE TRANSPORTER1 (PHT1)-type genes and PURPLE ACID PHOSPHATASE genes. PtoWRKY40 bound to the W box in the promoter of several PtoPHT1s and repressed their expression. Moreover, PtoWRKY40 interacted with PtoPHR1-LIKE3 (PtoPHL3), a PHR1 homolog in poplar, to inhibit the latter binding to the P1BS element and thus reduced PtoPHT1s' transcription under Pi-sufficient conditions. However, Pi deficiency decreased PtoWRKY40 abundance and therefore released its inhibition on PHT1s. In conclusion, we have uncovered a PSR mechanism mediated by PtoWRKY40 and PtoPHL3 which regulates Pi content in poplars, deepening our understanding of how poplars adapt to diverse Pi conditions and regulate appropriate responses to maintain Pi homeostasis.

Soybean responds to phosphate starvation through reversible protein phosphorylation

Phosphorus (P) deficiency is considered as a major constraint on crop production. Although a set of adaptative strategies are extensively suggested in soybean (Glycine max) to phosphate (Pi) deprivation, molecular mechanisms underlying reversible protein phosphorylation in soybean responses to P deficiency remains largely unclear. In this study, isobaric tags for relative and absolute quantitation, combined with liquid chromatography and tandem mass spectrometry analysis was performed to identify differential phosphoproteins in soybean roots under Pi sufficient and deficient conditions. A total of 427 phosphoproteins were found to exhibit differential accumulations, with 213 up-regulated and 214 down-regulated. Among them, a nitrate reductase, GmNR4 exhibiting increased phosphorylation levels under low Pi conditions, was further selected to evaluate the effects of phosphorylation on its nitrate reductase activity and subcellular localization. Mutations of GmNR4 phosphorylation levels significantly influenced its activity in vitro, but not for its subcellular localization. Taken together, identification of differential phosphoproteins reveled the complex regulatory pathways for soybean adaptation to Pi starvation through reversible protein phosphorylation.

Characterization of a complex involved in lipid transport to mitochondria during plant adaptation to phosphate starvation

Phosphate (Pi) starvation is a frequent nutrient stress encountered by plants to which they adapt by exerting different mechanisms. The partial degradation of phospholipids, a common constituent of cellular membranes, is a widespread response observed in plants to increase the intracellular Pi availability. To maintain membrane integrity, the degraded phospholipids are replaced by a non-phosphorous plastid-synthetized lipid, the digalactosyldiacylglycerol (DGDG). This replacement implies a lipid transport by unknown mechanisms from plastids to other organelles, such as mitochondria or plasma membrane. Precursor works in our laboratory have shown that DGDG transport to mitochondria occurs by non-vesicular transfer at plastids-mitochondria membrane contact sites (MCSs), the abundance of which increases during Pi starvation. Recently, we identified the mitochondrial transmembrane lipoprotein (MTL) complex, a huge complex enriched in lipids and mainly composed of proteins located in both mitochondrial membranes. One of its components of the inner membrane, AtMic60, was shown to play a role in intra-mitochondrial DGDG trafficking. Furthermore, the identification of plastidial proteins in the MTL complex suggests it might be located at mitochondria-plastids MCSs to promote lipid transport in response to Pi starvation. The MTL complex is also present in Pi-sufficient conditions and the structure of this huge complex is currently unknown. Thus, deciphering how the MTL structure and composition are modified in response to low Pi will be a key step to understand how this complex could be involved in MCSs formation and lipid transport and to identify new proteins involved in these processes. The goal of this work is to optimize a protocol to purify the MTL complex from Arabidopsis thaliana cell cultures grown in presence or absence of Pi and to analyze its structure and composition. To achieve our goal, we tested a combination of different fractionation methods to separate the MTL complex from other mitochondrial complexes: sucrose gradient, gel filtration and anion exchange chromatography. To date, the best results were obtained by using a combination of anion exchange and size exclusion chromatography. The first imaging attempts by negative stain electron microscopy and cryo-electron microscopy gave encouraging results concerning the homogeneity of our sample. Proteomic analyses of the purified complex are under progress and will help us to decipher whether further purification steps are required or not. To conclude, this preliminary work reveals that the MTL complex is resistant to a combination of chromatography techniques and paves the way for further characterizations of this complex. The new insights gained on the MTL complex will allow us to understand how lipids are transported to mitochondria during adaptation to Pi starvation and will contribute to the understanding of mitochondria MCSs formation in plants.

MYB-like transcription factor NoPSR1 is crucial for membrane lipid remodeling under phosphate starvation in the oleaginous microalga Nannochloropsis oceanica.

Membrane lipid remodeling under phosphate (Pi) limitation, a process that replaces structural membrane phospholipids with nonphosphorus lipids, is a widely observed adaptive response in plants and algae. Here, we identified the transcription factor phosphorus starvation response 1 (NoPSR1) as an indispensable player for regulating membrane lipid conversion during Pi starvation in the microalga Nannochloropsisoceanica. Knocking out NoPSR1 scarcely perturbed membrane lipid composition under Pi-sufficient conditions but significantly impaired dynamic alteration in membrane lipids during Pi starvation. In contrast, the absence of NoPSR1 led to no obvious change in cell proliferation or storage lipid accumulation under either nutrient-sufficient or Pi-deficient conditions. Our results demonstrate a key factor controlling the membrane lipid profile during the Pi starvation response in N.oceanica.

Effect of phytochrome-mediated red light signaling on phosphorus uptake and accumulation in rice

ABSTRACT Phosphorus (P) is a macronutrient essential for plant growth and productivity. Plants uptake P as inorganic phosphate (Pi); however, in the natural ecosystem, Pi availability is frequently a severe limiting factor for plant growth. Thus, plants have evolved several mechanisms, such as the expression of Pi starvation-responsive genes, to adapt to Pi deficient conditions. Although we recently reported that phytochrome (Phy)-mediated red light signaling promotes Pi uptake by increasing expression levels of such genes in the model plant Arabidopsis, it remains elusive whether a similar mechanism exists in agricultural crops. In the present study, we analyzed the effects of red light signaling on Pi uptake in rice (Oryza sativa L.) using osphyA and osphyB single mutants, and the osphyA osphyB double mutant. Unlike osphyA seedlings, osphyB seedlings showed a reduction in Pi uptake and Pi content. Furthermore, illumination of wild-type seedlings with red light significantly promoted Pi uptake, whereas illumination with blue or far-red light did not. The osphyB mutant showed reduced expression levels of several Pi starvation-responsive genes including Pi transporter genes. Additionally, these phenotypes of osphyB knockout mutants were much more evident under Pi deficient conditions than under Pi sufficient conditions. Moreover, red light promoted Pi uptake in seedlings of other plant species including broccoli (Brassica oleracea L. var. italica) and lettuce (Latuca sativa L.). These results suggest that OsPhyB-mediated red light signaling promotes Pi uptake in rice by up-regulating the expression of Pi starvation response-associated genes, and this phenomenon may be conserved in a wide range of plant species.

The ferroxidase LPR5 functions in the maintenance of phosphate homeostasis and is required for normal growth and development of rice.

Members of the Low Phosphate Root (LPR) family have been identified in rice (Oryza sativa) and expression analyses have been conducted. Here, we investigated the functions of one of the five members in rice, LPR5. qRT-PCR and promoter-GUS reporter analyses indicated that under Pi-sufficient conditions OsLPR5 was highly expressed in the roots, and specific expression occurred in the leaf collars and nodes, and its expression was increased under Pi-deficient conditions. In vitro analysis of the purified OsLPR5 protein showed that it exhibited ferroxidase activity. Overexpression of OsLPR5 triggered higher ferroxidase activity, and elevated concentrations of Fe(III) in the xylem sap and of total Fe in the roots and shoots. Transient expression of OsLPR5 in Nicotiana benthamiana provided evidence of its subcellular localization to the cell wall and endoplasmic reticulum. Knockout mutation in OsLPR5 by means of CRISPR-Cas9 resulted in adverse effects on Pi translocation, on the relative expression of Cis-NATOsPHO1;2, and on several morphological traits, including root development and yield potential. Our results indicate that ferroxidase-dependent OsLPR5 has both a broad-spectrum influence on growth and development in rice as well as affecting a subset of physiological and molecular traits that govern Pi homeostasis.

Modulation of Shoot Phosphate Level and Growth by PHOSPHATE1 Upstream Open Reading Frame.

Inorganic orthophosphate (Pi) is an essential nutrient for plant growth, and its availability strongly impacts crop yield. PHOSPHATE1 (PHO1) transfers Pi from root to shoot via Pi export into root xylem vessels. In this work, we demonstrate that an upstream open reading frame (uORF) present in the 5' untranslated region of the Arabidopsis (Arabidopsis thaliana) PHO1 inhibits its translation and influences Pi homeostasis. The presence of the uORF strongly inhibited the translation of a PHO1 5'UTR-luciferase construct in protoplasts. A point mutation removing the PHO1 uORF (ΔuORF) in transgenic Arabidopsis resulted in increased association of its mRNA with polysomes and led to higher PHO1 protein levels, independent of Pi availability. Interestingly, deletion of the uORF led to higher shoot Pi content and was associated with improved shoot growth under low external Pi supply and no deleterious effects under Pi-sufficient conditions. We further show that natural accessions lacking the PHO1 uORF exhibit higher PHO1 protein levels and shoot Pi content. Increased shoot Pi content was linked to the absence of the PHO1 uORF in a population of F2 segregants. We identified the PHO1 uORF in genomes of crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aesativum), and we verified the inhibitory effect of the rice PHO1 uORF on translation in protoplasts. Our work suggests that regulation of PHO1 expression via its uORF might be a genetic resource useful-both in natural populations and in the context of genome editing-toward improving plant growth under Pi-deficient conditions.

PROTEIN PHOSPHATASE95 Regulates Phosphate Homeostasis by Affecting Phosphate Transporter Trafficking in Rice.

Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (Oryza sativa), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing OsPP95 reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in ospp95 mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.