Study of the different regulation systems set up by the plant to optimize its phosphate absorption in the soil
The perception of ions by plants is a scientific challenge. Indeed, we are still far from mastering the functioning of these regulatory pathways, which remain among the least well characterised in photosynthetic organisms. This is probably due to their physiological importance, which has led to the redundancy of many compounds, thus hindering genetic approaches. For several years we have focused on the phosphate ion (Pi). Deficiency of this essential macroelement causes numerous physiological, developmental and biochemical changes in plants. In addition, it closely controls the production of numerous molecules with varied applications (medicines, energy production, etc.) and biomass. The CEA has developed numerous pioneering studies in the past based on the use of radiotracers that have made it possible to study the complexity of the movements of this ion in the soil and its absorption by plants. Thanks to a collaboration with Japanese colleagues, we have used radioisotope imaging to follow the absorption of Pi in real time and to show the little-known role of certain cellular bases in this process. This ion has the particularity of being particularly poorly mobile and present in limiting quantities in most soils. The study of its bioavailability is therefore particularly important. Indeed, the use of phosphate fertiliser to compensate for this problem is a necessity for agriculture, but this natural resource is limited and not very effective (only about 20% of the Pi applied is used by plants).
A/ Root tip where blue staining reveals cell nuclei. B/ Detail of cells where transcript spots are visible in green. C/ Visualisation of 33Pi entry into a root
Research to find alternatives to the large-scale spraying used so far is crucial to limit the associated pollution. In addition, the depletion of natural deposits will lead to the exploitation of lower quality resources contaminated by various toxic metals. This poses environmental and public health problems that will need to be addressed.
Due to the low availability of phosphate in the soil, plants have set up different regulatory systems to favour and optimise its uptake at the root level and its use in the whole plant. Our work suggests that most of the plant’s responses to deficiency are not the direct result of the metabolic consequences of phosphate deficiency. They are in fact linked to the detection of the quantities of phosphate present in the environment by specific regulatory pathways that we are seeking to elucidate.
Our laboratory is therefore interested in the different steps regulating phosphate homeostasis in the model plant Arabidopsis, at the cell, tissue and whole plant levels. In collaboration with various teams, we have conducted pioneering transcriptomic studies to characterise the global repercussions of Pi deficiency. We are studying Pi uptake and the various regulations affecting the high-affinity transporters of the PHT1 family as well as the importance of their subcellular localisation. Our most recent experiments have led us to develop real-time transcription imaging. This allows us to study the progression of Pi perception in each plant cell as well as the precise contribution of the many components we are trying to identify and characterise.
- Ried MK, Wild R, Zhu J, Pipercevic J, Sturm K, Broger L, Harmel RK, Abriata LA, Hothorn LA, Fiedler D, Hiller S, Hothorn M (2021) Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis. Nat Commun 12: 384
- Wei, P., Demulder, M., David, P., Eekhout, T., Yoshiyama, K., Okamoto; N.L., Vercauteren, I., Eeckhout, D., Galle, M., De Jaeger, G., Larsen, P., Audenaert, D., Desnos, T., Nussaume, L., Loris, R. and De Veylder, L. 2021. Arabidopsis casein kinase 2 triggers stem cell exhaustion under Al toxicity and phosphate deficiency through activating the DNA damage response pathway. Plant Cell, 33, 1361-1380.
- Blein, T., Balzergue, C., Roulé, T., Gabriel, M., Scalisi, L., François, T., Sorin, C., Christ, A., Godon, C., Delannoy, E., Martin-Magniette, M.-L., Nussaume, L., Hartmann, C., Gautheret, D., Desnos, T. and Crespi, M. 2020. Landscape of the non-coding transcriptome of Col and Ler Arabidopsis ecotypes in response to phosphate starvation. Plant Physiol. 183, 1058-73.
- Chevalier F., Cuyas L., Jouhet J., Gros V., Chiarenza S., Secco D., Whelan J., Seddiki K., Block M., Nussaume L., Maréchal E. (2019) Interplay between Jasmonic Acid, Phosphate Signaling and the Regulation of Glycerolipid Homeostasis in Arabidopsis. Plant and Cell Physiology, Oxford University Press, 2019. DOI: 10.1093/pcp/pcz027
- Zhu J, Lau K, Puschmann R, Harmel RK, Zhang Y, Pries V, Gaugler P, Broger L, Dutta AK, Jessen HJ, Schaaf G, Fernie AR, Hothorn LA, Fiedler D, Hothorn M (2019) Two bifunctional inositol pyrophosphate kinases/ phosphatases control plant phosphate homeostasis. Elife 8
- Godon, C., Mercier, C., Wang, X., David, P., Richaud, P., Nussaume, L., Liu, D., and Desnos, T. 2019. Under phosphate starvation conditions, Fe and Al trigger accumulation of the transcription factor STOP1 in the nucleus of Arabidopsis root cells. Plant J. 99, 937-949.
- Hanchi M., Thibaud M.C., Légeret B., Kuwata K., Pochon N., Beisson F., Cao A., Cuyas L., David P., Doerner P., Ferjani A, Li-Beisson Y, Mutterer J., Secco D., Lai F., Whelan J., Philibert M., Raghothama K.G., Rivasseau C., Nussaume L., Javot H.(2018). The phosphate fast-responsive genes PECP1 and PPsPase1 affect phosphocholine and phosphoethanolamine content. Plant Physiology. 176(4):2943-2962. DOI: 10.1104/pp.17.01246.
- Bonnot C., Nussaume L., Desnos T.(2018) Identification of Chemical Inducers of the Phosphate-Starvation Signaling Pathway in A. thaliana Using Chemical Genetics. Plant Chemical Genomics, 1795, Humana Press, 242 p., 2018, Methods in Molecular Biology, 978-1-4939-7874-8. DOI : 10.1007/978-1-4939-7874-8_6
- Balzergue, C., Dartevelle, T., Godon, C., Laugier, E., Meisrimler, C., Teulon, J.M., Creff, A., Bissler, M., Brouchoud, C., Hagège, A., Müller, J., Chiarenza, S., Péret, B., Delannoy, E., Javot, H., Thibaud, M.C., Armengaud, J., Abel, S., Pellequer, J.L., Nussaume, L. et Desnos, T. 2017. Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Nature communications 8; 15300, doi:10.1038/ncomms15300.
- Chevalier F., Carrera L.C., Nussaume L., Maréchal E. (2016). Chemical Genetics in Dissecting Membrane Glycerolipid Functions. Subcell. Biochem. 86, 159–175. DOI: 10.1007/978-3-319-25979-6_7
- Bonnot C., Pinson B., Clément M., Bernillon S., Chiarenza S., Kanno S., Kobayashi N., Delannoy E., Nakanishi T.M., Nussaume L., Desnos T.(2016) A chemical genetic strategy identify the PHOSTIN, a synthetic molecule that triggers phosphate starvation responses in Arabidopsis thaliana. New Phytologist., 209(1):161-176. DOI : 10.1111/nph.13591
- Kanno S., Cuyas L., Javot H., Bligny R., Gout E., Dartevelle T., Hanchi M., Nakanishi T.M., Thibaud M.-C., Nussaume L.(2016) Performance and Limitations of Phosphate Quantification: Guidelines for Plant Biologists. Plant and Cell Physiology, 57, 690–706. DOI: 10.1093/pcp/pcv208.
- Kanno S., Arrighi J.-F., Chiarenza S., Bayle V., Berthomé R., Péret B., Javot H., Delannoy E., Marin E., Nakanishi T.M., Thibaud M.-C., Nussaume L. (2016) A Novel Role for the Root Cap in Phosphate Uptake and Homeostasis. eLife, 5, e14577. DOI : 10.7554/eLife.14577.
- Cardona-López X., Cuyàs L., Marin E., Rajulu C., Irigoyen M.L., Gil E., Puga M.I., Bligny R., Nussaume L., Geldner N., Paz-Ares J. and Rubio V. (2015). ESCRT-III-associated protein AtALIX mediates high affinity phosphate transporter trafficking to maintain phosphate homeostasis in Arabidopsis. Plant Cell 27, 2560-2581
- Ayadi A., David P., Arrighi J.-F., Chiarenza S., Thibaud M.-C., Nussaume L., Marin E.(2015) Reducing the genetic redundancy of Arabidopsis PHT1 transporters to study phosphate uptake and signaling. Plant Physiology. 167: 1511 – 1526. DOI : 10.1104/pp.114.252338
- Secco D., Shou H., Schultz M.D., Chiarenza S., Nussaume L., Ecker J.R., Whelan J. and Lister R. (2015). Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements . eLife e09343.
- Péret B., Desnos T., Jost R., Kanno S., Berkowitz O., Nussaume L. (2014). Root architecture responses: in search for phosphate. Plant Physiol 166: 1713-1723.
- Arnaud C., Clément M., Thibaud M.-C. Javot H., Chiarenza S., Delannoy E., Revol J., Soreau P., Balzergue S., Block M.A., Maréchal E., Desnos T., 1,2,3 *, and Nussaume L. (2014). Identification of phosphatin, a drug alleviating Pi starvation responses in Arabidopsis. Plant Physiol 166: 1479-1491.
- Kanno S., Yamawaki, M., Ishibashi H., Kobayashi N. I. , Tanoi, K., Nussaume L. and Nakanishi T. M. (2012). Real-time Radioisotope Imaging System Developed for Plant Nutrient Uptake. Philosophical Transactions B 367,1501-8.
- Bayle V., Arrighi J.-F., Creff A., Nespoulous C., Vialaret J., Rossignol M., Gonzalez E., Paz-Ares J. and Nussaume L. (2011). Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of post-translational regulation. Plant Cell 23, 1523-35.
- Hirsch, J., Misson, J., Crisp, P.A., David, P., Bayle, V., Estavillo, G.M., Javot, H., Chiarenza, S., Mallory, A.C., Maizel, A., Declerck, M., Pogson, B.J., Vaucheret, H., Crespi, M., Desnos, T., Thibaud, M.-C., Nussaume, L., Marin, E. (2011) A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5′->3′ exoribonuclease (XRN) activities in Arabidopsis thaliana roots. PLoS ONE 6, e16724, 1-12.
- Thibaud M.C., Arrighi J.F., Bayle V., Chiarenza S., Creff A., Bustos R., Paz-Ares J., Poirier Y. and Nussaume L. (2010). Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J. 64, 775-789.
- Ticconi C.A., Lucero R.D., Sakhonwasee S., Adamson A.W., Creff A., Nussaume L., Desnos T., and S. Abel. (2009). ER-resident proteins, PDR2 and LPR1, mediate the developmental response ofroot meristems to phosphate availability. Proc. Natl. Acad. Sci. USA. 106, 14174-14179.
- Svistoonoff S., Creff A., Reymond M., Ricaud L., Blanchet A., Nussaume L. and Desnos T. (2007). Root tip contact with low-phosphate media reprogrammes plant root architecture. Nature Genetics, 39, 792-796.
- Hirsch J., Marin E., Floriani M., Chiarenza S., Richaud P., Nussaume L. and Thibaud MC. (2006), Phosphate deficiency promotes modification of iron distribution in Arabidopsis plants. Biochimie. 88, 1767-1771.
- Misson J., Raghothama K., Jain A., Jouhet J., Block M., Bligny R., Ortet P., Creff A., Somerville S., Rolland N., Doumas P., Nacry P., Herrerra-Estrella L., Nussaume L. and Thibaud M.-C. (2005) Transcriptional analysis using the Arabidopsis thaliana whole genome Affymetrix gene chips determined plants responses to phosphate deprivation. Proc. Natl. Acad. Sci. USA 102, 11934-9.
- Misson J., Thibaud M-C., Bechtold N., Raghotama K. and Nussaume L. (2004) Transcriptional regulation and functional properties of Arabidopsis Pht1 ;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants. Plant Mol Biol, 55, 727-741.
Crédit : N. Léonhardt – S. Chiarenza /CEA
To stay alive all cells need a tight control of their plasma membrane potential and proton gradients. In plants this is fulfilled by the plasma membrane H+-ATPases (AHA, 12 members in Arabidopsis). The importance of these transporters has been shown for many plant physiological functions including pH homeostasis, gaz and water exchanges with the environment, absorption of nutrients, hormone signaling. In the laboratory, to evaluate the function diversity and the regulation mechanisms of the AHA family, we concentrate our effort on two plant tissues where H+-ATPases play a crucial role: the guard cell, located in the epidermis of the leaves, which control carbon uptake and water loss with the atmosphere and the root.
Guard cells located in the epidermis of the leaves control carbon uptake and water loss with the atmosphere and present rapid changes in their turgor potential in response to various environmental parameters such as light, CO2 partial pressure, humidity, water stress and pathogen attacks. A loss of turgor from guard cells during a water stress is an essential process to limit transpiration at the epidermal level, avoiding plant dehydration. Guard cells also provide an ideal single cell system to dissect the functions of individual genes and proteins within signaling cascades. Interestingly, a limited number of AHA proteins are expressed in Arabidopsis guard cell facilitating genetics analyses. The functions of the major guard cell plasma membrane H+-ATPases isoforms are currently analyzed in details. In particular, we have shown that a down-regulation of the activity of these enzymes is a crucial step in ABA-induced stomatal closure and that AHA1, the most highly guard cell-expressed isoform, plays a crucial role in blue-light-induced stomatal opening and starch degradation. We investigate their ability to control the membrane potential and their regulation in response to changes in environmental conditions such as light/dark transition, drought stress and CO2. We also study the post-translational regulation of these proteins including the regulation by 14-3-3 proteins and phosphorylation events as previously done for other guard cell signaling components.
Schéma d’une cellule de garde – © N. Léonhardt
To identify new regulators involved in the control of proton pumps in guard cell, we performed a genetic screen using infrared thermal imaging Altogether, our work allows to decipher the functional diversity of proton pump in guard cell signalling and their regulation.
Mutants d’Arabidopsis – © N. Léonhardt
Role of proton pumps in roots
The role of proton pumps in the development and the architecture of the root system is also studied, as well as the nutrition and detoxification mechanisms of certain toxic elements.
The SAVE team is involved in the DEMETERRES project, funded by the Agence Nationale de la Recherche (ANR) as part of the RSNR call for Future Investments since 2013. Its ambition is to develop in France a set of innovative technologies for the remediation of contaminated soils and effluents, selective of radionucleides (mainly cesium137), non-intrusive and optimized in terms of secondary waste, which jointly affect the field of biotechnologies (bioremediation and phyto-extraction) and so-called eco-compatible physico-chemical technologies. This project, coordinated by the CEA/DRF, is structured around scientific expertise on physico-chemical and biological approaches, and the industrial know-how of the actors. It brings together CEA research teams at the DRF (BIAM in Cadarache) and the DEN (DPC, DTCD, DRCP and ICSM), the IRSN in Cadarache as well as INRA and CIRAD in Montpellier. The industrial partners are AREVA and VEOLIA.
The absorption of cesium by the plant depends on multiple parameters including the physico-chemical properties of the soil and physiological state of the plants. Since cesium is a weakly hydrated alkaline metal with chemical properties close to potassium, it is accepted that Cs enters plants principally via K transport systems devoted to plant nutrition. In addition, rhizospheric parameters at soil/root interface and their modification by the root system are crucial in Cs uptake. In particular, the root architecture, plasticity and transport activity can change soil pH and redox conditions affecting cesium bioavailability and its uptake by plants. Among the plant transporters, plasma membrane H+-ATPases are the major active transporters involved in ion uptake in roots via the regulation of membrane potential and play an important role by affecting the rhizosphere physicochemical properties. In this context, we investigate the impact of the proton-pumping ATPases activity in order to increase (phytoremediation) or limit (safe-food) cesium absorption by modulating Cs availability in the rhizosphere and/or by regulating root transport systems for Cs absorption.
Observation de l’extrusion des protons par les racines © N. Léonhardt / CEA
Contact : Nathalie PRAT
In many plant species, phosphate (Pi) deficiency modifies the architecture of the root system. This appears to be an adaptive response. Indeed, the induced architecture favours the exploration of superficial soil horizons rather than the deeper ones that are less rich in phosphate.
Our project focuses on the early growth arrest of the Arabidopsis primary root under Pi deficiency conditions. We have shown that this rapid growth arrest is iron and low pH dependent and correlates with epidermal cell wall hardening in the elongation zone, as measured by nanomechanical atomic force microscopy (AFM) (1).
Using classical genetics, we identified LPR1, STOP1 and ALMT1, major genes involved in this root growth arrest (1, 2, 3, 4). The LPR1 gene encodes a copper oxidase with ferroxidase activity (5). STOP1 is a C2H2-type zinc finger transcription factor that directly activates the expression of ALMT1, encoding a malate exudation transporter (1). STOP1 and ALMT1 were already known to play a role in tolerance to very low pH and aluminium (6, 7). We have recently shown that under low pH, iron and aluminium stimulate the accumulation of the STOP1 protein in the root cell nucleus (8). We are currently studying this regulatory step in particular.
© Caroline Mercier / CEA
Contact : Thierry DESNOS
- Balzergue C*, Dartevelle T*, Godon C*, Laugier E*, Meisrimler C*, Teulon JM, Creff A, Bissler M, Brouchoud C, Hagège A, Müller J, Chiarenza S, Javot H, Becuwe-Linka N, David P, Péret B, Delannoy E, Thibaud MC, Armengaud J, Abel S, Pellequer JL, Nussaume L, Desnos T. Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Commun. 8:15300 (2017).
- Reymond M., S. Svistoonoff, O. Loudet, L. Nussaume and T. Desnos. Identification of QTL controlling root growth response to phosphate starvation in Arabidopsis thaliana. Plant, Cell and Environment, 29:115-125 (2006).
- Svistoonoff S, Creff A, Reymond M, Sigoillot-Claude C, Ricaud L, Blanchet A, Nussaume L, Desnos T. Root tip contact with low-phosphate media reprogrammes plant root architecture. Nature Genetics, 39:792-796 (2007).
- Ticconi CA, Lucero RD, Sakhonwasee S, Adamson AW, Creff A, Nussaume L, Desnos T, Abel S. ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability. Proc Natl Acad Sci U S A. 106(33):14174-9 (2009).
- Müller J, Toev T, Heisters M, Teller J, Moore KL, Hause G, Dinesh DC, Bürstenbinder K, Abel S. Iron-dependent callose deposition adjusts root meristem maintenance to phosphate availability. Dev Cell. 20;33(2):216-30 (2015).
- Iuchi S, Koyama H, Iuchi A, Kobayashi Y, Kitabayashi S, Kobayashi Y, Ikka T, Hirayama T, Shinozaki K, Kobayashi M. Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Proc Natl Acad Sci U S A.104(23):9900-5 (2007).
- Hoekenga OA, Maron LG, Piñeros MA, Cançado GM, Shaff J, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Yamamoto Y, Koyama H, Kochian LV. AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci U S A. 103(25):9738-43 (2006).
- Godon C*, Mercier C*, Wang X, David P, Richaud P, Nussaume L, Liu D°, Desnos T. Under phosphate starvation condition, Fe and Al trigger the transcription factor STOP1 to accumulate in the nucleus of Arabidopsis root cells.Plant J. (in press).
Chloroplastes de la mousse Plagiomnium affine – Crédit : Fabelfroh
From the chloroplast to the nucleus: reactive oxygen species signalling and photo-oxidative stress in plants.
Under natural conditions, plants are often exposed to stresses due to, for example, variations in temperature, light or water availability. These stresses disturb the photosynthetic metabolism and lead to a high production of active oxygen species, especially singlet oxygen. At high concentrations, these species are toxic and result in an oxidative stress situation, associated with damage to macromolecules and a reduction in photosynthetic activity. One of our research projects aims to characterise the functions of antioxidant detoxification and repair systems implemented by plants in response to photo-oxidative stress.
The chloroplast contains a wide range of small lipid-soluble antioxidant molecules, such as carotenoids, tocopherols and other prenylated quinones, or water-soluble ones, such as ascorbate and pyrodoxin. Most of these antioxidants are singlet oxygen scavengers that function as vitamins in humans. We are investigating the protective functions of this network of chloroplast antioxidant molecules, in particular using knockout or over-expression mutants of the model plant Arabidopsis thaliana.
Imagerie d’autoluminescence du stress oxydant chez Arabidopsis.
© Michel Havaux / CEA
Signalling function of oxidation products
Active oxygen species (AOS) have a dual effect. In addition to their toxicity, AOS can function as signals capable of inducing changes in gene expression.
This dual role has recently been recognised for singlet oxygen formed in the chloroplast from excited chlorophyll molecules. Singlet oxygen signalling pathways can lead to either cell death or acclimatisation to oxidative stress.
We study the signalling of photo-oxidative stress in plants. In particular, we are trying to understand how the singlet oxygen signal is perceived in the chloroplast and transferred to the nucleus.
In this context, we are interested in the signalling function of oxidation products of preferential targets of singlet oxygen in the chloroplast such as carotenoids or lipids.
Arabidopsis plants facing volatile “signal” molecules in hermetic boxes.
© Michel Havau x/ CEA
Contact : Michel Havaux
Arabidopsis thaliana; Cell biology; Genetics; Metals; Stomata; Root Architecture racinaire; Transporters; Signal transduction; Phosphate; Plant Physiology; QTL; Growth; Root, Water stress