Visualizing plant intracellular inorganic orthophosphate distribution.

Intracellular inorganic orthophosphate (Pi) distribution and homeostasis profoundly affect plant growth and development. However, its distribution patterns remain elusive owing to the lack of efficient cellular Pi imaging methods. Here we develop a rapid colorimetric Pi imaging method, inorganic orthophosphate staining assay (IOSA), that can semi-quantitatively image intracellular Pi with high resolution. We used IOSA to reveal the alteration of cellular Pi distribution caused by Pi starvation or mutations that alter Pi homeostasis in two model plants, rice and Arabidopsis, and found that xylem parenchyma cells and basal node sieve tube element cells play a critical role in Pi homeostasis in rice. We also used IOSA to screen for mutants altered in cellular Pi homeostasis. From this, we have identified a novel cellular Pi distribution regulator, HPA1/PHO1;1, specifically expressed in the companion and xylem parenchyma cells regulating phloem Pi translocation from the leaf tip to the leaf base in rice. Taken together, IOSA provides a powerful method for visualizing cellular Pi distribution and facilitates the analysis of Pi signalling and homeostasis from the level of the cell to the whole plant.

Elevating plant immunity by translational regulation of a rice WRKY transcription factor.

Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquitin-proteasome system but was accumulated following elicitor or pathogen treatment, whereas the alternate product initiated from the downstream in-frame start codon was stable. Both the full and alternate OsWRKY7 proteins have transcriptional activities in yeast and rice cells, and overexpression of each form enhanced resistance to Xoo infection. Furthermore, disruption of the main AUG in rice increased the endogenous translation of the alternate stabilized form of OsWRKY7 and enhanced bacterial blight resistance. This study provides insights into the coordination of alternative translation and protein stability in the regulation of plant growth and basal defence mediated by the OsWRKY7 transcription factor, and also suggests a promising strategy to breed disease-resistant rice by translation initiation control.

The D14-SDEL1-SPX4 cascade integrates the strigolactone and phosphate signalling networks in rice.

Modern agriculture needs large quantities of phosphate (Pi) fertilisers to obtain high yields. Information on how plants sense and adapt to Pi is required to enhance phosphorus-use efficiency (PUE) and thereby promote agricultural sustainability. Here, we show that strigolactones (SLs) regulate rice root developmental and metabolic adaptations to low Pi, by promoting efficient Pi uptake and translocation from roots to shoots. Low Pi stress triggers the synthesis of SLs, which dissociate the Pi central signalling module of SPX domain-containing protein (SPX4) and PHOSPHATE STARVATION RESPONSE protein (PHR2), leading to the release of PHR2 into the nucleus and activating the expression of Pi-starvation-induced genes including Pi transporters. The SL synthetic analogue GR24 enhances the interaction between the SL receptor DWARF 14 (D14) and a RING-finger ubiquitin E3 ligase (SDEL1). The sdel mutants have a reduced response to Pi starvation relative to wild-type plants, leading to insensitive root adaptation to Pi. Also, SLs induce the degradation of SPX4 via forming the D14-SDEL1-SPX4 complex. Our findings reveal a novel mechanism underlying crosstalk between the SL and Pi signalling networks in response to Pi fluctuations, which will enable breeding of high-PUE crop plants.

NIGT1 represses plant growth and mitigates phosphate starvation signaling to balance the growth response tradeoff in rice.

Inorganic phosphate (Pi) availability is an important factor which affects the growth and yield of crops, thus an appropriate and effective response to Pi fluctuation is critical. However, how crops orchestrate Pi signaling and growth under Pi starvation conditions to optimize the growth defense tradeoff remains unclear. Here we show that a Pi starvation-induced transcription factor NIGT1 (NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1) controls plant growth and prevents a hyper-response to Pi starvation by directly repressing the expression of growth-related and Pi-signaling genes to achieve a balance between growth and response under a varying Pi environment. NIGT1 directly binds to the promoters of Pi starvation signaling marker genes, like IPS1, miR827, and SPX2, under Pi-deficient conditions to mitigate the Pi-starvation responsive (PSR). It also directly represses the expression of vacuolar Pi efflux transporter genes VPE1/2 to regulate plant Pi homeostasis. We further demonstrate that NIGT1 constrains shoot growth by repressing the expression of growth-related regulatory genes, including brassinolide signal transduction master regulator BZR1, cell division regulator CYCB1;1, and DNA replication regulator PSF3. Our findings reveal the function of NIGT1 in orchestrating plant growth and Pi starvation signaling, and also provide evidence that NIGT1 acts as a safeguard to avoid hyper-response during Pi starvation stress in rice.

A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana.

Nitrogen (N) deficiency causes early leaf senescence, resulting in accelerated whole-plant maturation and severely reduced crop yield. However, the molecular mechanisms underlying N-deficiency-induced early leaf senescence remain unclear, even in the model species Arabidopsis thaliana. In this study, we identified Growth, Development and Splicing 1 (GDS1), a previously reported transcription factor, as a new regulator of nitrate (NO3-) signaling by a yeast-one-hybrid screen using a NO3- enhancer fragment from the promoter of NRT2.1. We showed that GDS1 promotes NO3- signaling, absorption and assimilation by affecting the expression of multiple NO3- regulatory genes, including Nitrate Regulatory Gene2 (NRG2). Interestingly, we observed that gds1 mutants show early leaf senescence as well as reduced NO3- content and N uptake under N-deficient conditions. Further analyses indicated that GDS1 binds to the promoters of several senescence-related genes, including Phytochrome-Interacting Transcription Factors 4 and 5 (PIF4 and PIF5) and represses their expression. Interestingly, we found that N deficiency decreases GDS1 protein accumulation, and GDS1 could interact with Anaphase Promoting Complex Subunit 10 (APC10). Genetic and biochemical experiments demonstrated that Anaphase Promoting Complex or Cyclosome (APC/C) promotes the ubiquitination and degradation of GDS1 under N deficiency, resulting in loss of PIF4 and PIF5 repression and consequent early leaf senescence. Furthermore, we discovered that overexpression of GDS1 could delay leaf senescence and improve seed yield and N-use efficiency (NUE) in Arabidopsis. In summary, our study uncovers a molecular framework illustrating a new mechanism underlying low-N-induced early leaf senescence and provides potential targets for genetic improvement of crop varieties with increased yield and NUE.

Alternative splicing of REGULATOR OF LEAF INCLINATION 1 modulates phosphate starvation signaling and growth in plants.

Phosphate (Pi) limitation represents a primary constraint on crop production. To better cope with Pi deficiency stress, plants have evolved multiple adaptive mechanisms for phosphorus acquisition and utilization, including the alteration of growth and the activation of Pi starvation signaling. However, how these strategies are coordinated remains largely unknown. Here, we found that the alternative splicing (AS) of REGULATOR OF LEAF INCLINATION 1 (RLI1) in rice (Oryza sativa) produces two protein isoforms: RLI1a, containing MYB DNA binding domain and RLI1b, containing both MYB and coiled-coil (CC) domains. The absence of a CC domain in RLI1a enables it to activate broader target genes than RLI1b. RLI1a, but not RLI1b, regulates both brassinolide (BL) biosynthesis and signaling by directly activating BL-biosynthesis and signaling genes. Both RLI1a and RLI1b modulate Pi starvation signaling. RLI1 and PHOSPHATE STARVATION RESPONSE 2 function redundantly to regulate Pi starvation signaling and growth in response to Pi deficiency. Furthermore, the AS of RLI1-related genes to produce two isoforms for growth and Pi signaling is widely present in both dicots and monocots. Together, these findings indicate that the AS of RLI1 is an important and functionally conserved strategy to orchestrate Pi starvation signaling and growth to help plants adapt to Pi-limitation stress.

A reciprocal inhibitory module for Pi and iron signaling.

Phosphorous (P) and iron (Fe), two essential nutrients for plant growth and development, are highly abundant elements in the earth's crust but often display low availability to plants. Due to the ability to form insoluble complexes, the antagonistic interaction between P and Fe nutrition in plants has been noticed for decades. However, the underlying molecular mechanism modulating the signaling and homeostasis between them remains obscure. Here, we show that the possible iron sensors HRZs, the iron deficiency-induced E3 ligases, could interact with the central regulator of phosphate (Pi) signaling, PHR2, and prompt its ubiquitination at lysine residues K319 and K328, leading to its degradation in rice. Consistent with this, the hrzs mutants displayed a high Pi accumulation phenotype. Furthermore, we found that iron deficiency could attenuate Pi starvation signaling by inducing the expression of HRZs, which in turn trigger PHR2 protein degradation. Interestingly, on the other hand, rice PHRs could negatively regulate the expression of HRZs to modulate iron deficiency responses. Therefore, PHR2 and HRZs form a reciprocal inhibitory module to coordinate Pi and iron signaling and homeostasis in rice. Taken together, our results uncover a molecular link between Pi and iron master regulators, which fine-tunes plant adaptation to Pi and iron availability in rice.

A spatial-temporal understanding of gene regulatory networks and NtARF-mediated regulation of potassium accumulation in tobacco.

MAIN CONCLUSION: After tobacco topping, changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the NtARF genes, thus causing changes in K+ content. Tobacco (Nicotiana tabacum) is a valuable industrial and commercial crop, and the leaf is its primary product. Topping (removing apical buds) is a common agronomic practice that significantly improves the yield of tobacco leaves. Potassium (K+) plays an important physiological role in tobacco growth and leaf traits, including combustibility, aroma, and safety in cigarette products, and its levels are significantly decreased after topping. Here, to present global spatial-temporal gene expression profiles and gene regulatory networks of the core elements of K+ uptake, leaves and roots from topped and untopped plants at short- and long-term time points after topping were sampled for transcriptome analysis. We found that the wounding response was initiated in leaves in the early stages after topping. Then, in the long term, processes related to metabolism and transcription regulation, as well as ion binding and transport, were altered. The expression profiles showed that core elements of K+ uptake and xylem loading were drastically suppressed in roots after topping. Finally, transient expression experiments confirmed that changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the tobacco auxin response factor (NtARF) genes, thus causing changes in the K+ content. These results suggest that some ARFs could be selected as targets to enhance the expressions of K+ uptake transporters, leading to increment of K+ contents and improvement of leaf quality in tobacco breeding.

Two RING-Finger Ubiquitin E3 Ligases Regulate the Degradation of SPX4, An Internal Phosphate Sensor, for Phosphate Homeostasis and Signaling in Rice.

SPX-domain-containing proteins (SPXs) play an important role in inorganic phosphate (Pi) sensing, signaling, and transport in eukaryotes. In plants, SPXs are known to integrate cellular Pi status and negatively regulate the activity of Pi central regulators, the PHOSPATE STARVATION RESPONSE proteins (PHRs). The stability of SPXs, such as SPX4, is reduced under Pi-deficient conditions. However, the mechanisms by which SPXs are degraded remain unclear. In this study, using a yeast-two-hybrid screen we identified two RING-finger ubiquitin E3 ligases regulating SPX4 degradation, designated SDEL1 and SDEL2, which were post-transcriptionally induced by Pi starvation. We found that both SDELs were located in the nucleus and cytoplasm, had ubiquitin E3 ligase activity, and directly ubiquitinated the K213 and K299 lysine residues in SPX4 to regulate its stability. Furthermore, we found that PHR2, a Pi central regulator in rice, could compete with SDELs by interacting with SPX4 under Pi-sufficient conditions, which protected SPX4 from ubiquitination and degradation. Consistent with the biochemical function of SDEL1 and SDEL2, overexpression of SDEL1 or SDEL2 resulted in Pi overaccumulation and induced Pi-starvation signaling even under Pi-sufficient conditions. Conversely, their loss-of-function mutants displayed decreased Pi accumulation and reduced Pi-starvation signaling. Collectively, our study revealed that SDEL1 and SDEL2 facilitate the degradation of SPX4 to modulate PHR2 activity and regulate Pi homeostasis and Pi signaling in response to external Pi availability in rice.

Identification of vacuolar phosphate efflux transporters in land plants.

Inorganic phosphate (Pi) is an essential component of all life forms. Land plants acquire Pi from the soil through roots and associated symbioses, and it is then transported throughout the plant. When sufficient, excess Pi is stored in vacuoles for remobilization following Pi deficiency. Although Pi release from the vacuoles to the cytoplasm serves as a critical mechanism for plants to adapt to low-Pi stress, the transporters responsible for vacuolar Pi efflux have not been identified. Here, we identified a pair of Oryza sativa vacuolar Pi efflux transporters (OsVPE1 and OsVPE2) that were more abundant in plants grown under Pi-deficient conditions. These OsVPE proteins can transport Pi into yeast cells and Xenopus laevis oocytes. Vacuolar Pi content was higher in the loss-of-function Osvpe1 Osvpe2 double mutant than in wild type, particularly under low-Pi stress. Overexpression of either OsVPE1 or OsVPE2 in transgenic plants reduced vacuolar Pi content, consistent with a role in vacuolar Pi efflux. We demonstrate that these VPE proteins evolved from an ancient plasma membrane glycerol-3-phosphate transporter protein. Together, these data indicate that this transporter was recruited to the vacuolar membrane to catalyse Pi efflux during the course of land plant evolution.

An SPX-RLI1 Module Regulates Leaf Inclination in Response to Phosphate Availability in Rice.

Leaf erectness is one of the key traits of plant architecture; in grains, plants with upright leaves can be planted close together, thus benefiting yield/unit area. Many factors, such as hormones, affect leaf inclination; however, how nutrition status, in particular phosphate (Pi) availability, affects leaf inclination remains largely unexplained. Here, we show that in rice (Oryza sativa), Pi deficiency stress inhibits lamina joint cell elongation, thus restricting lamina joint size and inducing leaf erectness in rice. The Pi starvation-induced proteins SPX1 (for Syg1/Pho81/XPR1) and SPX2 play a negative role in the regulation of leaf inclination. We further identified an SPX1-interacting protein, REGULATOR OF LEAF INCLINATION1 (RLI1), which positively regulates leaf inclination by affecting lamina joint cell elongation in rice. The rli1 mutants showed reduced leaf inclination and the RLI1 overexpressors showed increased leaf inclination. RLI1 directly activates the downstream genes BRASSINOSTEROID UPREGULATED1 (BU1) and BU1-LIKE 1 COMPLEX1 to control elongation of the lamina joint cells, therefore enhancing leaf inclination. We also found that Pi deficiency repressed the expression of RLI1 SPX1 protein interacts directly with RLI1, which could prevent RLI1 binding to the promoters of downstream genes. Therefore, SPX and RLI1 form a module to regulate leaf inclination in response to external Pi availability in rice.

LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice.

Root system architecture is very important for plant growth and crop yield. It is essential for nutrient and water uptake, anchoring, and mechanical support. Root growth angle (RGA) is a vital constituent of root system architecture and is used as a parameter for variety evaluation in plant breeding. However, little is known about the underlying molecular mechanisms that determine root growth angle in rice (Oryza sativa). In this study, a rice mutant large root angle1 (lra1) was isolated and shown to exhibit a large RGA and reduced sensitivity to gravity. Genome resequencing and complementation assays identified OsPIN2 as the gene responsible for the mutant phenotypes. OsPIN2 was mainly expressed in roots and the base of shoots, and showed polar localization in the plasma membrane of root epidermal and cortex cells. OsPIN2 was shown to play an important role in mediating root gravitropic responses in rice and was essential for plants to produce normal RGAs. Taken together, our findings suggest that OsPIN2 plays an important role in root gravitropic responses and determining the root system architecture in rice by affecting polar auxin transport in the root tip.

ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice.

Root meristem activity determines root growth and root architecture and consequently affects water and nutrient uptake in plants. However, our knowledge about the regulation of root meristem activity in crop plants is very limited. Here, we report the isolation and characterization of a short root mutant in rice (Oryza sativa) with reduced root meristem activity. This root growth defect is caused by a mutation in ABNORMAL INFLORESCENCE MERISTEM1 (AIM1), which encodes a 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in ß-oxidation. The reduced root meristem activity of aim1 results from reduced salicylic acid (SA) levels and can be rescued by SA application. Furthermore, reduced SA levels are associated with reduced levels of reactive oxygen species (ROS) in aim1, likely due to increased expression of redox and ROS-scavenging-related genes, whose increased expression is (at least in part) caused by reduced expression of the SA-inducible transcriptional repressors WRKY62 and WRKY76. Like SA, ROS application substantially increased root length and root meristem activity in aim1 These results suggest that AIM1 is required for root growth in rice due to its critical role in SA biosynthesis: SA maintains root meristem activity through promoting ROS accumulation by inducing the activity of WRKY transcriptional repressors, which repress the expression of redox and ROS-scavenging genes.

Phosphate starvation induced OsPHR4 mediates Pi-signaling and homeostasis in rice.

KEY MESSAGE: OsPHR4 mediates the regulation of Pi-starvation signaling and Pi-homeostasis in a PHR1-subfamily dependent manner in rice. Phosphate (Pi) starvation response is a sophisticated process for plant in the natural environment. In this process, PHOSPHATE STARVATION RESPONSE 1 (PHR1) subfamily genes play a central role in regulating Pi-starvation signaling and Pi-homeostasis. Besides the three PHR1 orthologs in Oryza sativa L. (Os) [(Os) PHR1, (Os) PHR2, and (Os) PHR3], which were reported to regulated Pi-starvation signaling and Pi-homeostasis redundantly, a close related PHR1 ortholog [designated as (Os) PHR4] is presented in rice genome with unknown function. In this study, we found that OsPHR4 is a Pi-starvation induced gene and mainly expresses in vascular tissues through all growth and development periods. The expression of OsPHR4 is positively regulated by OsPHR1, OsPHR2 and OsPHR3. The nuclear located OsPHR4 can respectively interact with other three PHR1 subfamily members to regulate downstream Pi-starvation induced genes. Consistent with the positive role of PHR4 in regulating Pi-starvation signaling, the OsPHR4 overexpressors display higher Pi accumulation in the shoot and elevated expression of Pi-starvation induced genes under Pi-sufficient condition. Besides, moderate growth retardation and repression of the Pi-starvation signaling in the OsPHR4 RNA interfering (RNAi) transgenic lines can be observed under Pi-deficient condition. Together, we propose that OsPHR4 mediates the regulation of Pi-starvation signaling and Pi-homeostasis in a PHR1-subfamily dependent manner in rice.

Integrative Comparison of the Role of the PHOSPHATE RESPONSE1 Subfamily in Phosphate Signaling and Homeostasis in Rice.

Phosphorus (P), an essential macronutrient for all living cells, is indispensable for agricultural production. Although Arabidopsis (Arabidopsis thaliana) PHOSPHATE RESPONSE1 (PHR1) and its orthologs in other species have been shown to function in transcriptional regulation of phosphate (Pi) signaling and Pi homeostasis, an integrative comparison of PHR1-related proteins in rice (Oryza sativa) has not previously been reported. Here, we identified functional redundancy among three PHR1 orthologs in rice (OsPHR1, OsPHR2, and OsPHR3) using phylogenetic and mutation analysis. OsPHR3 in conjunction with OsPHR1 and OsPHR2 function in transcriptional activation of most Pi starvation-induced genes. Loss-of-function mutations in any one of these transcription factors (TFs) impaired root hair growth (primarily root hair elongation). However, these three TFs showed differences in DNA binding affinities and messenger RNA expression patterns in different tissues and growth stages, and transcriptomic analysis revealed differential effects on Pi starvation-induced gene expression of single mutants of the three TFs, indicating some degree of functional diversification. Overexpression of genes encoding any of these TFs resulted in partial constitutive activation of Pi starvation response and led to Pi accumulation in the shoot. Furthermore, unlike OsPHR2-overexpressing lines, which exhibited growth retardation under normal or Pi-deficient conditions, OsPHR3-overexpressing plants exhibited significant tolerance to low-Pi stress but normal growth rates under normal Pi conditions, suggesting that OsPHR3 would be useful for molecular breeding to improve Pi uptake/use efficiency under Pi-deficient conditions. We propose that OsPHR1, OsPHR2, and OsPHR3 form a network and play diverse roles in regulating Pi signaling and homeostasis in rice.

Genetic manipulation of a high-affinity PHR1 target cis-element to improve phosphorous uptake in Oryza sativa L.

Phosphorus (P) is an essential macronutrient for crop development and production. Phosphate starvation response 1 (PHR1) acts as the central regulator for Pi-signaling and Pi-homeostasis in plants by binding to the cis-element PHR1 binding sequence (P1BS; GNATATNC). However, how phosphate starvation-induced gene expression is regulated remains obscure. In this work, we investigated the DNA binding affinity of the PHR1 ortholog OsPHR2 to its downstream target genes in Oryza sativa (rice). We confirmed that a combination of P1BS and P1BS-like motifs are essential for stable binding by OsPHR2. Furthermore, we report that variations in P1BS motif bases affected the binding affinity of OsPHR2 and that the highest affinity motif was GaATATtC (designated the A-T-type P1BS). We also found that a combination of two A-T-type P1BS elements in tandem, namely HA-P1BS, was very efficient for binding of OsPHR2. Using the cis-regulator HA-P1BS, we modified the promoters of Transporter Traffic Facilitator 1 (PHF1), a key factor controlling endoplasmic reticulum-exit of phosphate transporters to the plasma membrane, for efficient uptake of phosphorous in an energetically neutral way. Transgenic plants with the modified promoters showed significantly enhanced tolerance to low phosphate stress in both solution and soil conditions, which provides a new strategy for crop improvement to enhance tolerance of nutrient deficiency.

Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner.

In plants, sensing the levels of external and internal nutrients is essential for reprogramming the transcriptome and adapting to the fluctuating environment. Phosphate (Pi) is a key plant nutrient, and a large proportion of Pi starvation-responsive genes are under the control of Phosphate Starvation Response Regulator 1 (PHR1) in Arabidopsis (AtPHR1) and its homologs, such as Oryza sativa (Os)PHR2 in rice. AtPHR1 and OsPHR2 expression is not very responsive to Pi starvation, raising the question as to how plants sense changes in cellular Pi levels to activate the central regulator. SPX [named after SYG1 (suppressor of yeast gpa1), Pho81 (CDK inhibitor in yeast PHO pathway), and XPR1 (xenotropic and polytropic retrovirus receptor)] proteins that harbor only the SPX domain are reported to be involved in the negative regulation of Pi starvation responses. Here, we show that the nuclear localized SPX proteins SPX1 and SPX2 are Pi-dependent inhibitors of the activity of OsPHR2 in rice. Indeed, SPX1 and SPX2 proteins interact with PHR2 through their SPX domain, inhibiting its binding to P1BS (the PHR1-binding sequence: GNATATNC). In vivo data, as well as results from in vitro experiments using purified SPX1, SPX2, and OsPHR2 proteins, showed that SPX1 and SPX2 inhibition of OsPHR2 activity is Pi-dependent. These data provide evidence to support the involvement of SPX1 and SPX2 in the Pi-sensing mechanism in plants.