Growing on calcareous soils and facing climate change.

Soil calcium carbonate (CaCO3) impacts plant mineral nutrition far beyond Fe metabolism, imposing constraints for crop growth and quality in calcareous agrosystems. Our knowledge on plant strategies to tolerate CaCO3 effects mainly refers to Fe acquisition. This review provides an update on plant cellular and molecular mechanisms recently described to counteract the negative effects of CaCO3 in soils, as well as recent efforts to identify genetic bases involved in CaCO3 tolerance from natural populations, that could be exploited to breed CaCO3-tolerant crops. Finally, we review the impact of environmental factors (soil water content, air CO2, and temperature) affecting soil CaCO3 equilibrium and plant tolerance to calcareous soils, and we propose strategies for improvement in the context of climate change.

The tonoplast localized protein PtNPF1 participates in the regulation of nitrogen response in diatoms.

Diatoms are a highly successful group of phytoplankton, well adapted also to oligotrophic environments and capable of handling nutrient fluctuations in the ocean, particularly nitrate. The presence of a large vacuole is an important trait contributing to their adaptive features. It confers diatoms the ability to accumulate and store nutrients, such as nitrate, when they are abundant outside and then to reallocate them into the cytosol to meet deficiencies, in a process called luxury uptake. The molecular mechanisms that regulate these nitrate fluxes are still not known in diatoms. In this work, we provide new insights into the function of Phaeodactylum tricornutum NPF1, a putative low-affinity nitrate transporter. To accomplish this, we generated overexpressing strains and CRISPR/Cas9 loss-of-function mutants. Microscopy observations confirmed predictions that PtNPF1 is localized on the vacuole membrane. Furthermore, functional characterizations performed on knock-out mutants revealed a transient growth delay phenotype linked to altered nitrate uptake. Together, these results allowed us to hypothesize that PtNPF1 is presumably involved in modulating intracellular nitrogen fluxes, managing intracellular nutrient availability. This ability might allow diatoms to fine-tune the assimilation, storage and reallocation of nitrate, conferring them a strong advantage in oligotrophic environments.

AraDiv: a dataset of functional traits and leaf hyperspectral reflectance of Arabidopsis thaliana.

Data from functional trait databases have been increasingly used to address questions related to plant diversity and trait-environment relationships. However, such databases provide intraspecific data that combine individual records obtained from distinct populations at different sites and, hence, environmental conditions. This prevents distinguishing sources of variation (e.g., genetic-based variation vs. phenotypic plasticity), a necessary condition to test for adaptive processes and other determinants of plant phenotypic diversity. Consequently, individual traits measured under common growing conditions and encompassing within-species variation across the occupied geographic range have the potential to leverage trait databases with valuable data for functional and evolutionary ecology. Here, we recorded 16 functional traits and leaf hyperspectral reflectance (NIRS) data for 721 widely distributed Arabidopsis thaliana natural accessions grown in a common garden experiment. These data records, together with meteorological variables obtained during the experiment, were assembled to create the AraDiv dataset. AraDiv is a comprehensive dataset of A. thaliana's intraspecific variability that can be explored to address questions at the interface of genetics and ecology.

Farnesoid X receptor alpha ligands inhibit HDV in vitro replication and virion infectivity.

BACKGROUND AND AIMS: HDV, a satellite of HBV, is responsible for the most severe form of human viral hepatitis, for which curative therapy is still awaited. Both HBV and HDV use the hepatic transporter of bile acids (ie, Na+-taurocholate cotransporting polypeptide) to enter hepatocytes. We have previously shown that ligands of the farnesoid-X-receptor alpha (FXR), a master regulator of bile acids metabolism, inhibit HBV replication. Here we asked whether FXR ligands can also control HDV infection. APPROACH AND RESULTS: In vitro HDV monoinfections or HDV/HBV coinfections and superinfections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes. Following treatment with FXR ligands, HDV RNAs and antigens were analyzed by RT-qPCR, northern blot, immunofluorescence, and western blot. Virus secretion was studied by RNA quantification in supernatants, and the infectivity of secreted HDV particles was measured by reinfection of naive HuH7.5-Na+-taurocholate cotransporting polypeptide cells. In HDV/HBV superinfection models, a 10-day treatment with FXR ligand GW4064 decreased intracellular HDV RNAs by 60% and 40% in dHepaRG cells and primary human hepatocytes, respectively. Both HDV genomic and antigenomic RNAs were affected by treatment, which also reduced the amount of intracellular delta antigen. This antiviral effect was also observed in HDV monoinfected dHepaRG cells, abolished by FXR loss of function, and reproduced with other FXR ligands. In HBV/HDV coinfected dHepaRG cells, HDV secretion was decreased by 60% and virion-specific infectivity by >95%. CONCLUSIONS: FXR ligands both inhibit directly (ie, independently of anti-HBV activity) and indirectly (ie, dependently of anti-HBV activity) the replication, secretion, and infectivity of HDV. The overall anti-HDV activity was superior to that obtained with interferon-α, highlighting the therapeutic potential of FXR ligands in HDV-infected patients.

ARSK1 activates TORC1 signaling to adjust growth to phosphate availability in Arabidopsis.

Nutrient sensing and signaling are essential for adjusting growth and development to available resources. Deprivation of the essential mineral phosphorus (P) inhibits root growth.1 The molecular processes that sense P limitation to trigger early root growth inhibition are not known yet. Target of rapamycin (TOR) kinase is a central regulatory hub in eukaryotes to adapt growth to internal and external nutritional cues.2,3 How nutritional signals are transduced to TOR to control plant growth remains unclear. Here, we identify Arabidopsis-root-specific kinase 1 (ARSK1), which attenuates initial root growth inhibition in response to P limitation. We demonstrate that ARSK1 phosphorylates and stabilizes the regulatory-associated protein of TOR 1B (RAPTOR1B), a component of the TOR complex 1, to adjust root growth to P availability. These findings uncover signaling components acting upstream of TOR to balance growth to P availability.

Molecular framework integrating nitrate sensing in root and auxin-guided shoot adaptive responses.

Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs.

A Perspective on Plant Phenomics: Coupling Deep Learning and Near-Infrared Spectroscopy.

The trait-based approach in plant ecology aims at understanding and classifying the diversity of ecological strategies by comparing plant morphology and physiology across organisms. The major drawback of the approach is that the time and financial cost of measuring the traits on many individuals and environments can be prohibitive. We show that combining near-infrared spectroscopy (NIRS) with deep learning resolves this limitation by quickly, non-destructively, and accurately measuring a suite of traits, including plant morphology, chemistry, and metabolism. Such an approach also allows to position plants within the well-known CSR triangle that depicts the diversity of plant ecological strategies. The processing of NIRS through deep learning identifies the effect of growth conditions on trait values, an issue that plagues traditional statistical approaches. Together, the coupling of NIRS and deep learning is a promising high-throughput approach to capture a range of ecological information on plant diversity and functioning and can accelerate the creation of extensive trait databases.

Mineral nutrient signaling controls photosynthesis: focus on iron deficiency-induced chlorosis.

Photosynthetic organisms convert light energy into chemical energy stored in carbohydrates. To perform this process, an adequate supply of essential mineral elements, such as iron, is required in the chloroplast. Because iron plays a crucial role during electron transport and chlorophyll formation, iron deficiency alters photosynthesis and promotes chlorosis, or the yellowing of leaves. Intriguingly, iron deficiency-induced chlorosis can be reverted by the depletion of other micronutrients [i.e., manganese (Mn)] or macronutrients [i.e., sulfur (S) or phosphorus (P)], raising the question of how plants integrate nutrient status to control photosynthesis. Here, we review how improving our understanding of the complex relationship between nutrient homeostasis and photosynthesis has great potential for crop improvement.

The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23.

Nitrate and potassium nutrition is tightly coordinated in vascular plants. Physiological and molecular genetics studies have demonstrated that several NPF/NRT1 nitrate transporters have a significant impact on both uptake and the root-shoot partition of these nutrients. However, how these traits are biochemically connected remain controversial since some NPF proteins, e.g. NPF7.3/NRT1.5, have been suggested to mediate K+/H+ exchange instead of nitrate fluxes. Here we show that NPF6.2/NRT1.4, a protein that gates nitrate accumulation at the leaf petiole of Arabidopsis thaliana, also affects the root/shoot distribution of potassium. We demonstrate that NPF6.2/NRT1.4 is a plasma membrane nitrate transporter phosphorylated at threonine-98 by the CIPK23 protein kinase that is a regulatory hub for nitrogen and potassium nutrition. Heterologous expression of NPF6.2/NRT1.4 and NPF7.3/NRT1.5 in yeast mutants with altered potassium uptake and efflux systems showed no evidence of nitrate-dependent potassium transport by these proteins.

GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways.

Plants need to cope with strong variations of nitrogen availability in the soil. Although many molecular players are being discovered concerning how plants perceive NO3- provision, it is less clear how plants recognize a lack of nitrogen. Following nitrogen removal, plants activate their nitrogen starvation response (NSR), which is characterized by the activation of very high-affinity nitrate transport systems (NRT2.4 and NRT2.5) and other sentinel genes involved in N remobilization such as GDH3. Using a combination of functional genomics via transcription factor perturbation and molecular physiology studies, we show that the transcription factors belonging to the HHO subfamily are important regulators of NSR through two potential mechanisms. First, HHOs directly repress the high-affinity nitrate transporters, NRT2.4 and NRT2.5. hho mutants display increased high-affinity nitrate transport activity, opening up promising perspectives for biotechnological applications. Second, we show that reactive oxygen species (ROS) are important to control NSR in wild-type plants and that HRS1 and HHO1 overexpressors and mutants are affected in their ROS content, defining a potential feed-forward branch of the signaling pathway. Taken together, our results define the relationships of two types of molecular players controlling the NSR, namely ROS and the HHO transcription factors. This work (i) up opens perspectives on a poorly understood nutrient-related signaling pathway and (ii) defines targets for molecular breeding of plants with enhanced NO3- uptake.

The XPB Subunit of the TFIIH Complex Plays a Critical Role in HIV-1 Transcription and XPB Inhibition by Spironolactone Prevents HIV-1 Reactivation from Latency.

HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH, and concurrently suppresses acute HIV infection in vitro Here we investigated SP as a possible block-and-lock agent for a functional cure aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals upon cell stimulation with latency reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock HIV cure approaches.IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual's HIV loads to below the detection limit, nevertheless rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level virus expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency. We investigated the potential of repurposing the FDA-approved spironolactone (SP), as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.

Sugar and Nitrate Sensing: A Multi-Billion-Year Story.

Sugars and nitrate play a major role in providing carbon and nitrogen in plants. Understanding how plants sense these nutrients is crucial, most notably for crop improvement. The mechanisms underlying sugar and nitrate sensing are complex and involve moonlighting proteins such as the nitrate transporter NRT1.1/NFP6.3 or the glycolytic enzyme HXK1. Major components of nutrient signaling, such as SnRK1, TOR, and HXK1, are relatively well conserved across eukaryotes, and the diversification of components such as the NRT1 family and the SWEET sugar transporters correlates with plant terrestrialization. In plants, Tre6P plays a hormone-like role in plant development. In addition, nutrient signaling has evolved to interact with the more recent hormone signaling, allowing fine-tuning of physiological and developmental responses.

Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes.

Few proteins have been characterized as abscisic acid transporters. Several of them are NRT1/PRT Family (NPF) transporters which have been characterized in yeast using reporter systems. Because several members of the NPF4 subfamily members were identified in yeast as ABA transporters, here, we screened for ABA transport activity the seven members of the NPF4 subfamily in Xenopus oocytes using cRNA injection and 3H-ABA accumulation. The ABA transport capacities of NPF4.2, NPF4.5, NPF4.6, and NPF4.7 were confirmed. The transport properties of NPF4.5 and NPF4.6 were studied in more detail. Both ABA transporter activities are pH-dependent and slightly pH-dependent apparent Km around 500 µM. There is no competitive inhibition of the ABA-analogs pyrabactin and quinabactin on ABA accumulation demonstrating a different selectivity compared to the ABA receptors. Functional expression of these ABA transporters in Xenopus oocyte is an opportunity to start structure-function studies and also to identify partner proteins of these hormone transporters.

Human T-Cell Lymphotropic Virus Type 1 Transactivator Tax Exploits the XPB Subunit of TFIIH during Viral Transcription.

Human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein is required for viral gene expression. Tax transactivates the viral promoter by recruiting specific transcription factors but also by interfering with general transcription factors involved in the preinitiation step, such as TFIIA and TFIID. However, data are lacking regarding Tax interplay with TFIIH, which intervenes during the last step of preinitiation. We previously reported that XPB, the TFIIH subunit responsible for promoter opening and promoter escape, is required for Tat-induced human-immunodeficiency virus promoter transactivation. Here, we investigated whether XPB may also play a role in HTLV-1 transcription. We report that Tax and XPB directly interact in vitro and that endogenous XPB produced by HTLV-1-infected T cells binds to Tax and is recruited on proviral LTRs. In contrast, XPB recruitment at the LTR is not detected in Tax-negative HTLV-1-infected T cells and is strongly reduced when Tax-induced HTLV-1 LTR transactivation is blocked. XPB overexpression does not affect basal HTLV-1 promoter activation but enhances Tax-mediated transactivation in T cells. Conversely, downregulating XPB strongly reduces Tax-mediated transactivation. Importantly, spironolactone (SP)-mediated inhibition of LTR activation can be rescued by overexpressing XPB but not XPD, another TFIIH subunit. Furthermore, an XPB mutant defective for the ATPase activity responsible for promoter opening does not show rescue of the effect of SP. Finally, XPB downregulation reduces viability of Tax-positive but not Tax-negative HTLV-1-transformed T cell lines. These findings reveal that XPB is a novel cellular cofactor hijacked by Tax to facilitate HTLV-1 transcription.IMPORTANCE HTLV-1 is considered the most potent human oncovirus and is also responsible for severe inflammatory disorders. HTLV-1 transcription is undertaken by RNA polymerase II and is controlled by the viral oncoprotein Tax. Tax transactivates the viral promoter first via the recruitment of CREB and its cofactors to the long terminal repeat (LTR). However, how Tax controls subsequent steps of the transcription process remains unclear. In this study, we explore the link between Tax and the XPB subunit of TFIIH that governs, via its ATPase activity, the promoter-opening step of transcription. We demonstrate that XPB is a novel physical and functional partner of Tax, recruited on HTLV-1 LTR, and required for viral transcription. These findings extend the mechanism of Tax transactivation to the recruitment of TFIIH and reinforce the link between XPB and transactivator-induced viral transcription.

Disruption of the Lotus japonicus transporter LjNPF2.9 increases shoot biomass and nitrate content without affecting symbiotic performances.

BACKGROUND: After uptake from soil into the root tissue, distribution and allocation of nitrate throughout the whole plant body, is a critical step of nitrogen use efficiency (NUE) and for modulation of plant growth in response to various environmental conditions. In legume plants nitrate distribution is also important for the regulation of the nodulation process that allows to fix atmospheric N (N2) through the symbiotic interaction with rhizobia (symbiotic nitrogen fixation, SNF). RESULTS: Here we report the functional characterization of the Lotus japonicus gene LjNPF2.9, which is expressed mainly in the root vascular structures, a key localization for the control of nitrate allocation throughout the plant body. LjNPF2.9 expression in Xenopus laevis oocytes induces 15NO3 accumulation indicating that it functions as a nitrate importer. The phenotypic characterization of three independent knock out mutants indicates an increased shoot biomass in the mutant backgrounds. This phenotype is associated to an increased/decreased nitrate content detected in the shoots/roots. Furthermore, our analysis indicates that the accumulation of nitrate in the shoot does not affect the nodulation and N-Fixation capacities of the knock out mutants. CONCLUSIONS: This study shows that LjNPF2.9 plays a crucial role in the downward transport of nitrate to roots, occurring likely through a xylem-to-phloem loading-mediated activity. The increase of the shoot biomass and nitrate accumulation might represent a relevant phenotype in the perspective of an improved NUE and this is further reinforced in legume plants by the reported lack of effects on the SNF efficiency.

Getting to the Root of Plant Mineral Nutrition: Combinatorial Nutrient Stresses Reveal Emergent Properties.

In nature, plants have to handle daily fluctuations in light and temperature. In addition, plants face biotic and abiotic stresses that often come in various combinations. For instance, the availability of various nutrients in soil is heterogeneous, resulting in combined nutrient stress. Recent studies reveal that plant responses to multiple nutrient stresses are not the summation of the plant responses to each individual stress. Here, we present and discuss the interactions between phosphate, nitrogen, and zinc to illustrate the effect of macro- and micronutrient interactions on plant growth and ion homeostasis. Solving the mystery of these interactions will pave the way to the development of strategies to improve crop productivity.

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.

Farnesoid X receptor-α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Responses to Systemic Nitrogen Signaling in Arabidopsis Roots Involve trans-Zeatin in Shoots.

Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N deprivation.