Identification and interest of molecular markers to monitor plant Pi status.

BACKGROUND: Inorganic phosphate (Pi) is the sole source of phosphorus for plants. It is a limiting factor for plant yield in most soils worldwide. Due to economic and environmental constraints, the use of Pi fertilizer is and will be more and more limited. Unfortunately, evaluation of Pi bioavailability or Pi starvation traits remains a tedious task, which often does not inform us about the real Pi plant status. RESULTS: Here, we identified by transcriptomic studies carried out in the plant model Arabidopsis thaliana, early roots- or leaves-conserved molecular markers for Pi starvation, exhibiting fast response to modifications of phosphate nutritional status. We identified their homologues in three crops (wheat, rapeseed, and maize) and demonstrated that they offer a reliable opportunity to monitor the actual plant internal Pi status. They turn out to be very sensitive in the concentration range of 0-50 µM which is the most common case in the vast majority of soils and situations where Pi hardly accumulates in plants. Besides in vitro conditions, they could also be validated for plants growing in the greenhouse or in open field conditions. CONCLUSION: These markers provide valuable physiological tools for plant physiologists and breeders to assess phosphate bio-availability impact on plant growth in their studies. This also offers the opportunity to cope with the rising economical (shortage) and societal problems (pollution) resulting from the management of this critical natural resource.

Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor.

STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by Western blot analysis in our sumoylation assay in Escherichia coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.

Arabidopsis ALIX Regulates Stomatal Aperture and Turnover of Abscisic Acid Receptors.

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

Interplay between Jasmonic Acid, Phosphate Signaling and the Regulation of Glycerolipid Homeostasis in Arabidopsis.

Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of glycerolipid remodeling in response to Pi deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and glycerolipid remodeling in wild-type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine, phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi deprivation, JA seems rather associated with a specific MGDG increase. Following Pi resupply, whereas the expression of glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, that is, downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content.

Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3.

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.

Performance and Limitations of Phosphate Quantification: Guidelines for Plant Biologists.

Phosphate (Pi) is a macronutrient that is essential for plant life. Several regulatory components involved in Pi homeostasis have been identified, revealing a very high complexity at the cellular and subcellular levels. Determining the Pi content in plants is crucial to understanding this regulation, and short real-time(33)Pi uptake imaging experiments have shown Pi movement to be highly dynamic. Furthermore, gene modulation by Pi is finely controlled by localization of this ion at the tissue as well as the cellular and subcellular levels. Deciphering these regulations requires access to and quantification of the Pi pool in the various plant compartments. This review presents the different techniques available to measure, visualize and trace Pi in plants, with a discussion of the future prospects.

Live single-cell transcriptional dynamics via RNA labelling during the phosphate response in plants.

Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.