PHOSPHATE2 Research Articles

AmPHO2 gene expression and interaction protein screening of snapdragon inoculated with AM fungi under low phosphorus stress

Phosphorus (P) is involved in flowering and fruiting in plants. P deficiency inhibits the formation of plant reproductive organs and affects the process of flower bud differentiation, leads to delayed flowering and affects the number and quality of flowers. Our previous experiment showed that arbuscular mycorrhizal (AM) fungal inoculation could promote snapdragon (Antirrhinum majus L.) uptaking P, prolong the flowering period, and increase the number of flower buds and flowers at the full bloom stage under low P stress. The aim of this study is to preliminarily explore the molecular mechanism of AM fungus enhancing P absorption by snapdragon under low P stress. Four treatments were set up in this experiment, namely, low P level (0.1 mmol /L P), low P level (0.1 mmol /L P) +AM fungi, normal P level (0.7 mmol /L P), and normal P level (0.7 mmol/L P) + AM fungi, to evaluate regulation of AM fungi on the phosphate signaling factor PHOSPHATE 2 (PHO2) and the snapdragon flowering. Results showed that the P content in the roots, shoot apical meristem, buds and flowers of snapdragon inoculated with AM fungi increased and the expression of AmPHO2 decreased, suggesting that mycorrhizal symbiosis may inhibit the expression of AmPHO2. Total 28 AmPHO2 interacting proteins were screened by Yeast two-hybrid technique, among which the thioredoxin AmTrx was involved in the flowering process of the plant, and point-to-point validation and dual luciferase assay of AmTrx was further verified. This study showed that inoculation with AM fungi could negatively regulate AmPHO2, promote P uptake, flowering, and improve flower quality of snapdragon under low P stress.

Physiological traits and expression profile of genes associated with nitrogen and phosphorous use efficiency in wheat

Nitrogen (N) and phosphorous (P) play a very important role in the growth and development of wheat as well as major constituents of biological membranes. To meet the plant's nutritional demand these nutrients are applied in the form of fertilizers. But the plant can utilize only half of the applied fertilizer whereas the rest is lost through surface runoff, leaching and volatilization. Thus, to overcome the N/P loss we need to elucidate the molecular mechanism behind the N/P uptake. In our study, we used DBW16 (low NUE), and WH147 (high NUE) wheat genotypes under different doses of N, whereas HD2967 (low PUE) and WH1100 (high PUE) genotypes were studied under different doses of P. To check the effect of different doses of N/P, the physiological parameters like total chlorophyll content, net photosynthetic rate, N/P content, and N/PUE of these genotypes were calculated. In addition, gene expression of various genes involved in N uptake, utilization, and acquisition such as Nitrite reductase (NiR), Nitrate transporter 1/Peptide transporter family (NPF2.4/2.5), Nitrate transporter (NRT1) and NIN Like Protein (NLP) and induced phosphate starvation (IPS), Phosphate Transporter (PHT1.7) and Phosphate 2 (PHO2) acquisition was studied by quantitative real-time PCR. Statistical analysis revealed a lower percent reduction in TCC, NPR, and N/P content in N/P efficient wheat genotypes (WH147 & WH1100). A significant increase in relative fold expression of genes under low N/P concentration was observed in N/P efficient genotypes as compared to N/P deficient genotypes. Significant differences in physiological data and gene expression among N/ P efficient and deficient wheat genotypes could be useful for future improvement of N/P use efficiency.

Whole-Transcriptome Analysis Reveals Long Noncoding RNAs Involved in Female Floral Development of Hickory (Carya cathayensis Sarg.).

Hickory, an endemic woody oil and fruit tree species in China, is of great economic value. However, hickory has a long juvenile period and an inconsistent flowering of males and females, thus influencing the bearing rates and further limiting fruits yield. Currently, it is reported that long noncoding RNAs (lncRNAs) play critical regulatory roles in biological processes. However, the role of lncRNAs in the development of hickory female flowers remains unclear. In this study, a total of 6,862 putative lncRNAs were identified from the female flower transcriptomes in three different growth stages of hickory. We proposed that lncRNAs might play an important role in phytohormone signaling processes for flower formation, especially in the abscisic acid and jasmonic acid pathways, according to the results of our Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Moreover, we predicted the interactions among four microRNAs (miRNAs), three lncRNAs, and four genes. We proposed that facing the changing environment, LNC_002115 competes with PHOSPHATE2 (PHO2) for the binding sites on cca-miR399f, and protects PHO2 from suppression. In addition, cis-acting LNC_002115 regulates the expression of the SHORT VEGETATIVE PHASE (SVP) by influencing ABRE-binding factor (ABF). In brief, LNC_002115 regulates hickory female floral development by impacting both PHO2 and SVP. This study was the first to identify lncRNAs involved in hickory female floral development, and provided new insight to elucidate how lncRNAs and their targets play a role in female floral development in hickory, thus unfolding the opportunities for functional characterization of blossom-related lncRNAs in further studies.

Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.

In this article, we describe a set of novel alfalfa (Medicago sativa L.) plants that hyper-accumulate Phosphate ion (Pi) at levels 3- to 6-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 encodes a ubiquitin conjugating E2 enzyme (UBC24) previously characterized in Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify PHO2 haplotypes in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in both PHO2-like genes (PHO2-B and PHO2-C). After delivery of the reagent and regeneration from transformed leaf explants, plants with mutations in all haplotypes of PHO2-B and PHO2-C were identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplotypes and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization for future alfalfa functional genomic studies.

Phosphorus homeostasis: acquisition, sensing, and long-distance signaling in plants.

Phosphorus (P), an essential nutrient required by plants often becomes the limiting factor for plant growth and development. Plants employ various mechanisms to sense the continuously changing P content in the soil. Transcription factors, such as SHORT ROOT (SHR), AUXIN RESPONSE FACTOR19 (ARF19), and ETHYLENE-INSENSITIVE3 (EIN3) regulate the growth of primary roots, root hairs, and lateral roots under low P. Crop improvement strategies under low P depend either on improving P acquisition efficiency or increasing P utilization. The various phosphate transporters (PTs) are involved in the uptake and transport of P from the soil to various plant cellular organelles. A plethora of regulatory elements including transcription factors, microRNAs and several proteins play a critical role in the regulation of coordinated cellular P homeostasis. Among these, the well-established P starvation signaling pathway comprising of central transcriptional factor phosphate starvation response (PHR), microRNA399 (miR399) as a long-distance signal molecule, and PHOSPHATE 2 (PHO2), an E2 ubiquitin conjugase is crucial in the regulation of phosphorus starvation responsive genes. Under PHR control, several classes of PHTs, microRNAs, and proteins modulate root architecture, and metabolic processes to enable plants to adapt to low P. Even though sucrose and inositol phosphates are known to influence the phosphorus starvation response genes, the exact mechanism of regulation is still unclear. In this review, a basic understanding of P homeostasis under low P in plants and all the above aspects are discussed.

Phosphate-induced resistance to pathogen infection in Arabidopsis.

SUMMARYIn nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss‐of‐function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)‐ and jasmonic acid (JA)‐dependent signaling pathways were higher in high Pi plants compared to wild‐type plants grown under control conditions, which is consistent with increased levels of SA and JA in non‐infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses.

Differentially expressed microRNAs that target functional genes in mature soybean nodules.

MicroRNAs (miRNAs) are important regulators of biological functions in plants. To find out what roles miRNAs play in regulating symbiotic nitrogen fixation (SNF) in soybean [Glycine max (L.) Merr.], we identified high-confidence differentially expressed (DE) miRNAs from uninoculated roots (UR), rhizobium-inoculated roots (IR), and nodules (NODs) of soybean by robust small RNA sequencing (sRNA-seq). Based on their predicted target messenger RNAs (mRNAs), the expression profiles of some of these DE miRNAs could be linked to nodule functions. In particular, several miRNAs associated with nutrient transportation genes were differentially expressed in IRs and mature NODs. MiR399b, specifically, was highly induced in IRs and NODs, as well as by inorganic phosphate (Pi) starvation. In composite soybean plants overexpressing miR399b, PHOSPHATE2 (PHO2), a known target of miR399b that inhibits the activities of high-affinity Pi transporters, was strongly repressed. In addition, the overexpression of miR399b in the roots of transgenic composite plants significantly improved whole-plant Pi and ureide concentrations and the overall growth in terms of leaf node numbers and whole-plant dry weight. Our findings suggest that the induction of miR399b in NODs could enhance nitrogen fixation and soybean growth, possibly via improving Pi uptake to achieve a better Pi-nitrogen balance to promote SNF in nodules.

Molecular Manipulation of the miR399/PHO2 Expression Module Alters the Salt Stress Response of Arabidopsis thaliana.

In Arabidopsis thaliana (Arabidopsis), the microRNA399 (miR399)/PHOSPHATE2 (PHO2) expression module is central to the response of Arabidopsis to phosphate (PO4) stress. In addition, miR399 has been demonstrated to also alter in abundance in response to salt stress. We therefore used a molecular modification approach to alter miR399 abundance to investigate the requirement of altered miR399 abundance in Arabidopsis in response to salt stress. The generated transformant lines, MIM399 and MIR399 plants, with reduced and elevated miR399 abundance respectively, displayed differences in their phenotypic and physiological response to those of wild-type Arabidopsis (Col-0) plants following exposure to a 7-day period of salt stress. However, at the molecular level, elevated miR399 abundance, and therefore, altered PHO2 target gene expression in salt-stressed Col-0, MIM399 and MIR399 plants, resulted in significant changes to the expression level of the two PO4 transporter genes, PHOSPHATE TRANSPORTER1;4 (PHT1;4) and PHT1;9. Elevated PHT1;4 and PHT1;9 PO4 transporter levels in salt stressed Arabidopsis would enhance PO4 translocation from the root to the shoot tissue which would supply additional levels of this precious cellular resource that could be utilized by the aerial tissues of salt stressed Arabidopsis to either maintain essential biological processes or to mount an adaptive response to salt stress.

Dissecting MicroRNA-mRNA Regulatory Networks Underlying Sulfur Assimilation and Cadmium Accumulation in Poplar Leaves.

The process of cadmium (Cd) accumulation and detoxification under different sulfur levels remains largely unknown in woody plants. To investigate the physiological and transcriptomic regulation mechanisms of poplars in response to different sulfate (S) supply levels and Cd exposure, we exposed Populus deltoides saplings to one of the low, moderate and high S levels together with either 0 or 50 µM Cd. Cd accumulation was decreased in low S-treated poplar leaves, and it tended to be increased in high S-supplied leaves under the Cd exposure condition. Sulfur nutrition was deficient in low S-supplied poplars, and it was improved in high S-treated leaves. Cd exposure resulted in lower sulfur level in the leaves supplied with moderate S, it exacerbated a Cd-induced sulfur decrease in low S-treated leaves and it caused a higher sulfur concentration in high S-supplied leaves. In line with the physiological changes, a number of mRNAs and microRNAs (miRNAs) involved in Cd accumulation and sulfur assimilation were identified and the miRNA-mRNA networks were dissected. In the networks, miR395 and miR399 members were identified as hub miRNAs and their targets were ATP sulfurylase 3 (ATPS3) and phosphate 2 (PHO2), respectively. These results suggest that Cd accumulation and sulfur assimilation are constrained by low and enhanced by high S supply, and Cd toxicity is aggravated by low and relieved by high S in poplar leaves, and that miRNA-mRNA regulatory networks play pivotal roles in sulfur-mediated Cd accumulation and detoxification in Cd-exposed poplars.

Genome-Wide Identification and Characterization of Hexokinase Genes in Moso Bamboo (Phyllostachys edulis).

Plant hexokinases (HXKs) are a class of multifunctional proteins that not only act as the enzymes required for hexose phosphorylation but also serve as sugar sensors that repress the expression of some photosynthetic genes when internal glucose level increases and regulators of cell metabolism and some sugar-related signaling pathways independent on their catalytic actives. The HXKs have been studied in many plants; however, limited information is available on HXKs of moso bamboo (Phyllostachys edulis). In this study, we identified and characterized 12 hexokinase genes in moso bamboo. Phylogenetic analysis revealed that the moso bamboo hexokinases (PeHXKs) were classifiable into five subfamilies which represented the three types of hexokinases in plants. Gene structure and conserved motif analysis showed that the PeHXK genes contained diverse numbers of introns and exons and that the encoded proteins showed similar motif organization within each subfamily. Multiple sequence alignment revealed that the PeHXK proteins contained conserved domains, such as phosphate 1 (P1), phosphate 2 (P2), adenosine, and a sugar-binding domain. Evolutionary divergence analysis indicated that the PeHXK, OsHXK, and BdHXK families underwent negative selection and experienced a large-scale duplication event approximately 19–319 million years ago. Expression analysis of the PeHXK genes in the leaf, stem, root, and rhizome of moso bamboo seedlings indicated that the PeHXKs perform pivotal functions in the development of moso bamboo. A protein subcellular localization assay showed that PeHXK5a, PeHXK8, and PeHXK3b were predominantly localized in mitochondria, and PeHXK8 protein was also detected in the nucleus. The HXK activity of the PeHXK5a, PeHXK8, and PeHXK3b was verified by a functional complementation assay using the HXK-deficient triple-mutant yeast strain YSH7.4-3C (hxk1, hxk2, and glk1), and the results showed that the three PeHXKs had the plant HXK-specific enzyme traits. The present findings would provide a foundation for further functional analysis of the PeHXK gene family.

CASEIN KINASE2-Dependent Phosphorylation of PHOSPHATE2 Fine-tunes Phosphate Homeostasis in Rice.

Plants have evolved complex physiological and biochemical mechanisms to adapt to a heterogeneous soil phosphorus environment. PHOSPHATE2 (PHO2) is a phosphate (Pi) starvation-signaling regulator involved in maintaining Pi homeostasis in plants. Arabidopsis (Arabidopsis thaliana) PHO2 targets PHOSPHATE TRANSPORTER1 (PHT1) and PHO1 for degradation, whereas rice (Oryza sativa) PHO2 is thought to mediate PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 degradation. However, it is unclear whether and how PHO2 is post-translationally regulated. Here, we show that in rice, the CASEIN KINASE2 (OsCK2) catalytic subunit OsCK2α3 interacts with OsPHO2 in vitro and in vivo in vascular tissues cells, and phosphorylates OsPHO2 at Ser-841. Phosphorylated OsPHO2 is degraded more rapidly than native OsPHO2 in cell-free degradation assays. OsPHO2 interacts with OsPHO1 and targets it for degradation through a multivesicular body-mediated pathway. PHO1 mutation partially rescued the pho2 mutant phenotype. Further genetic analysis showed that a nonphosphorylatable version of OsPHO2 rescued the Ospho2 phenotype of high Pi accumulation in leaves better than native OsPHO2. In addition to the previously established role of OsCK2 in negatively regulating endoplasmic reticulum exit of PHT1 phosphate transporters, this work uncovers a role for OsCK2α3 in modulating Pi homeostasis through regulating the phosphorylation status and abundance of OsPHO2 in rice.

Characterization and functional analysis of microRNA399 in Cunninghamia lanceolata

The miR399 is a conserved microRNA (miRNA) family, and it has been characterized as an essential regulator of phosphorus transport in plants. However, the biological function of miR399 in Cunninghamia lanceolata is still largely unclear. In this study, the comparison of mature miR399 sequence revealed a high similarity between Arabidopsis thaliana and C. lanceolate, and the pre-miR399 was capable of forming a typical stem-loop hairpin structure. A gene PHOSPHATE 2 (PHO2) was identified as a target of cln-miR399 using 5' rapid amplification of cDNA ends. Furthermore, the relationship between cln-miR399 and PHO2 was further confirmed through a transient co-expression of both genes in Nicotiana benthamiana. To examine the function of miR399 in Arabidopsis, miR399-overexpressing transgenic Arabidopsis thaliana was acquired using Agrobacterium-mediated approach. Real-time PCR showed that the amount of cln-MIR399 transcripts was higher in miR399-overexpressing plants than in wild-type plants, which was accompanied with down-regulation of expression of its target gene AtPHO2. The P content was 1.40 to 1.56-fold higher in the leaves of three transgenic lines than in wild type plants. However, the P content in the roots of the three transgenic lines was 24.5 - 37.2 % less than that in wild type plants. Moreover, the transcriptions of three phosphate transporter genes (PHT1, PHT2, and PHT3) were up-regulated in roots of miR399-overexpressing Arabidopsis plants. Interestingly, the transgenic lines exhibited retarded growth under normal P conditions compared with the wild type. Our findings demonstrate that cln-miR399 may play crucial roles in P transport and plant growth via regulation of its target gene PHO2.

Systematic Analysis of Alkaline/Neutral Invertase Genes Reveals the Involvement of Smi-miR399 in Regulation of SmNINV3 and SmNINV4 in Salvia miltiorrhiza.

Alkaline/neutral invertases (NINVs), which irreversibly catalyze the hydrolysis of sucrose into fructose and glucose, play crucial roles in carbohydrate metabolism and plant development. Comprehensive insights into NINV genes are lacking in Salvia miltiorrhiza, a well-known traditional Chinese medicinal (TCM) plant with significant medicinal and economic value. Through genome-wide prediction, nine putative SmNINV genes, termed SmNINV1-SmNINV9, were identified. Integrated analysis of gene structures, sequence features, conserved domains, conserved motifs and phylogenetic trees revealed the conservation and divergence of SmNINVs. The identified SmNINVs were differentially expressed in roots, stems, leaves, flowers, and different root tissues. They also responded to drought, salicylic acid, yeast extract, and methyl jasmonate treatments. More importantly, computational prediction and experimental validation showed that SmNINV3 and SmNINV4 were targets of Smi-miR399, a conserved miRNA previously shown to affect Pi uptake and translocation through the cleavage of PHOSPHATE2 (PHO2). Consistently, analysis of 43 NINV genes and 26 miR399 sequences from Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, and Solanum lycopersicum showed that various AtNINV, PtNINV, MeNINV, and SlNINV genes were regulated by miR399. It indicates that the miR399-NINV module exists widely in plants. Furthermore, Smi-miR399 also cleaved SmPHO2 transcripts in S. miltiorrhiza, suggesting the complexity of NINVs, PHO2, and miR399 networks.

DRB1, DRB2 and DRB4 Are Required for Appropriate Regulation of the microRNA399/PHOSPHATE2 Expression Module in Arabidopsis thaliana.

Adequate phosphorous (P) is essential to plant cells to ensure normal plant growth and development. Therefore, plants employ elegant mechanisms to regulate P abundance across their developmentally distinct tissues. One such mechanism is PHOSPHATE2 (PHO2)-directed ubiquitin-mediated degradation of a cohort of phosphate (PO4) transporters. PHO2 is itself under tight regulation by the PO4 responsive microRNA (miRNA), miR399. The DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2 and DRB4, have each been assigned a specific functional role in the Arabidopsis thaliana (Arabidopsis) miRNA pathway. Here, we assessed the requirement of DRB1, DRB2 and DRB4 to regulate the miR399/PHO2 expression module under PO4 starvations conditions. Via the phenotypic and molecular assessment of the knockout mutant plant lines, drb1, drb2 and drb4, we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in Arabidopsis shoot and root tissues; (2) DRB1 is the primary DRB required for miR399 production; (3) DRB2 and DRB4 play secondary roles in regulating miR399 production, and; (4) miR399 appears to direct expression regulation of the PHO2 transcript via both an mRNA cleavage and translational repression mode of RNA silencing. Together, the hierarchical contribution of DRB1, DRB2 and DRB4 demonstrated here to be required for the appropriate regulation of the miR399/PHO2 expression module identifies the extreme importance of P homeostasis maintenance in Arabidopsis to ensure that numerous vital cellular processes are maintained across Arabidopsis tissues under a changing cellular environment.

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants.

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.

The genetic architecture of phosphorus efficiency in sorghum involves pleiotropic QTL for root morphology and grain yield under low phosphorus availability in the soil

BackgroundPhosphorus (P) fixation on aluminum (Al) and iron (Fe) oxides in soil clays restricts P availability for crops cultivated on highly weathered tropical soils, which are common in developing countries. Hence, P deficiency becomes a major obstacle for global food security. We used multi-trait quantitative trait loci (QTL) mapping to study the genetic architecture of P efficiency and to explore the importance of root traits on sorghum grain yield on a tropical low-P soil.ResultsP acquisition efficiency was the most important component of P efficiency, and both traits were highly correlated with grain yield under low P availability. Root surface area was positively associated with grain yield. The guinea parent, SC283, contributed 58% of all favorable alleles detected by single-trait mapping. Multi-trait mapping detected 14 grain yield and/or root morphology QTLs. Tightly linked or pleiotropic QTL underlying the surface area of fine roots (1–2 mm in diameter) and grain yield were detected at positions 1–7 megabase pairs (Mb) and 71 Mb on chromosome 3, respectively, and a root diameter/grain yield QTL was detected at 7 Mb on chromosome 7. All these QTLs were near sorghum homologs of the rice serine/threonine kinase, OsPSTOL1. The SbPSTOL1 genes on chromosome 3, Sb03g006765 at 7 Mb and Sb03g031690 at 60 Mb were more highly expressed in SC283, which donated the favorable alleles at all QTLs found nearby SbPSTOL1 genes. The Al tolerance gene, SbMATE, may also influence a grain yield QTL on chromosome 3. Another PSTOL1-like gene, Sb07g02840, appears to enhance grain yield via small increases in root diameter. Co-localization analyses suggested a role for other genes, such as a sorghum homolog of the Arabidopsis ubiquitin-conjugating E2 enzyme, phosphate 2 (PHO2), on grain yield advantage conferred by the elite parent, BR007 allele.ConclusionsGenetic determinants conferring higher root surface area and slight increases in fine root diameter may favor P uptake, thereby enhancing grain yield under low-P availability in the soil. Molecular markers for SbPSTOL1 genes and for QTL increasing grain yield by non-root morphology-based mechanisms hold promise in breeding strategies aimed at developing sorghum cultivars adapted to low-P soils.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

Root Cell-Specific Regulators of Phosphate-Dependent Growth.

Cellular specialization in abiotic stress responses is an important regulatory feature driving plant acclimation. Our in silico approach of iterative coexpression, interaction, and enrichment analyses predicted root cell-specific regulators of phosphate starvation response networks in Arabidopsis (Arabidopsis thaliana). This included three uncharacterized genes termed Phosphate starvation-induced gene interacting Root Cell Enriched (PRCE1, PRCE2, and PRCE3). Root cell-specific enrichment of 12 candidates was confirmed in promoter-GFP lines. T-DNA insertion lines of 11 genes showed changes in phosphate status and growth responses to phosphate availability compared with the wild type. Some mutants (cbl1, cipk2, prce3, and wdd1) displayed strong biomass gain irrespective of phosphate supply, while others (cipk14, mfs1, prce1, prce2, and s6k2) were able to sustain growth under low phosphate supply better than the wild type. Notably, root or shoot phosphate accumulation did not strictly correlate with organ growth. Mutant response patterns markedly differed from those of master regulators of phosphate homeostasis, PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHOSPHATE2 (PHO2), demonstrating that negative growth responses in the latter can be overcome when cell-specific regulators are targeted. RNA sequencing analysis highlighted the transcriptomic plasticity in these mutants and revealed PHR1-dependent and -independent regulatory circuits with gene coexpression profiles that were highly correlated to the quantified physiological traits. The results demonstrate how in silico prediction of cell-specific, stress-responsive genes uncovers key regulators and how their manipulation can have positive impacts on plant growth under abiotic stress.

Knock out of the PHOSPHATE 2 Gene TaPHO2-A1 Improves Phosphorus Uptake and Grain Yield under Low Phosphorus Conditions in Common Wheat.

MiR399 and its target PHOSPHATE2 (PHO2) play pivotal roles in phosphate signaling in plants. Loss of function mutation in PHO2 leads to excessive Pi accumulation in shoots and growth retardation in diploid plants like Arabidopsis thaliana and rice (Oryza sativa). Here we isolated three PHO2 homologous genes TaPHO2-A1, -B1 and -D1 from hexaploid wheat (Triticum aestivum). These TaPHO2 genes all contained miR399-binding sites and were able to be degraded by tae-miR399. TaPHO2-D1 was expressed much more abundantly than TaPHO2-A1 and -B1. The ion beam-induced deletion mutants were used to analyze the effects of TaPHO2s on phosphorus uptake and plant growth. The tapho2-a1, tapho2-b1 and tapho2-d1 mutants all had significant higher leaf Pi concentrations than did the wild type, with tapho2-d1 having the strongest effect, and tapho2-b1 the weakest. Two consecutive field experiments showed that knocking out TaPHO2-D1 reduced plant height and grain yield under both low and high phosphorus conditions. However, knocking out TaPHO2-A1 significantly increased phosphorus uptake and grain yield under low phosphorus conditions, with no adverse effect on grain yield under high phosphorus conditions. Our results indicated that TaPHO2s involved in phosphorus uptake and translocation, and molecular engineering TaPHO2 shows potential in improving wheat yield with less phosphorus fertilizer.

Spatio-Temporal Transcript Profiling of Rice Roots and Shoots in Response to Phosphate Starvation and Recovery

Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation-induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.