Group leader: Juan Carlos del Pozo Benito - Research Professor INIA

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In the past century we has witnessed an extraordinary success of plant agronomy and breeding, resulting in the doubling of crop productivity. This increase was to a great extent the result of increasing inputs, water and fertilizers, pesticides and also genetic breeding. Present demographic tendencies, which predict a doubling of the population at 2050, and the growing interest to exploit plants as renewable energy sources are prompting us to augment crop production gains. However, nowadays, the circumstances are rather different: society is demanding an increase of the production but within a more sustainable agricultural system, increasing the yield with less inputs to make it maintainable and adaptable to the future generations. The plant root system, in addition to provide anchor to the soil and to establish the symbiotic relations with the rizosphere, is responsible of acquiring nutrients and water from the soil. In this context, the root system plays an important role in plant development and growth, and therefore, root system improvement, which has not been completely exploited, will be necessary to fulfill the future needs of the increasing human population in terms of food production.

Our main research areas are:

One of the challenges in our lab is to identify new genes and natural compounds that improve the growth of the root system under low nutrient availability, specifically under low phosphate (Pi) or low nitrate (N), two macronutrients whose shortage severely limits agricultural production. On the other hand, excess fertilization, in addition of the high cost to farmers, causes severe groundwater contamination, damaging the environment. One of the problems/limitations in root biology study in the last years has been that plants are grown in many cases with the root system in presence of light, what conditioned growth and responses. To identify root variability and novel genes associated with the response to a particular environment, we are using a new cultivation system called D-ROOT (Figure 1), which allow us to cultivate plants with the root system in darkness while shoot grows in a normal photoperiodic illumination, simulating natural condition (Silva-Navas et al., 2015).


Figure 1: The Dark-Root (D-Root) device allows to grow Arabidopsis seedlings in vitro with the root system in the darkness while exposing the shoot to the light.

a) Graphic representation of the D-Root device. b) The petri dish is inserted into the methacrylate box. Arrow points to the methacrylate combo used to block partially the light coming from the top. c) D-Root device adaptation to illuminate roots with different light wavelengths using led technology or a Uv-B lamp.



Using this system we have shown that root light avoidance (root growth opposite to the light focus) is mediated by flavonols. This compounds also acts as integrating molecules in the root meristem to balance the proliferating and differentiating pathway (auxin/cytokinin and H2O2/O2-) and to stablish a correct zonation in the root tip (Figure 2) (Silva-Navas et al., 2016).


Figure 2: Model of transition between cell division and differentiation in dark grown root meristems and in response to light.

(A)Flavonols regulate root zonation. Flavonols accumulate in cells undergoing differentiation repressing proliferation and promoting differentiation. Cytokinin-SHY2 and H2O2-UPB1 induce biosynthesis of flavonols to reduce cell proliferation through inhibition of PIN1, auxin polar transport and by scavenging superoxide anion (O2-). Auxin can trigger O2-production which is dependent on flavonol content. These mutual interactions define the boundary between cell proliferation and cell differentiation (dashed line). As light induces flavonols accumulation, the system is perturbed when roots are exposed to light, stimulus that reduces meristem size along with root growth.

(B) Avoidance of light by roots requires flavonols. Lighting of roots from one side induces flavonol accumulation at that side promoting differentiation and growth away from the spotlight. Dashed line indicates temporal movement of the boundary between cell proliferation and differentiation responsible of the light tropism.



Another project in the lab is focused in understand the role of BiAux, a novel metabolite identified specifically in roots. We have been able to synthesis BiAux in the lab, and application of this compound to plants (Arabidopsis or tomato) significantly increased the size of the root system, mainly by generating more and larger lateral roots. BiAux treatment increases expression the SKP2B::GUS, a lateral root marker, and DR5::GUS, an auxin response marker (Figure 3).  Preliminary results seems to indicate that BiAux acts as a modulator of the auxin receptor sensitivity, mainly of TIR1, AFB1 and AFB3 co-receptors. At present, we are exploring the molecular mode of action of BiAux by protein-ligand modelling and directed mutagenesis of co-receptors. 



Figure 3: BiAux increases lateral root formation and induces lateral root and auxin response markers. 


In addition, using the D-Root device, we have found that root responses to Pi starvation is significantly different to previously described by several groups (Figure 4). This differences prompted us to study this response in dark-grown roots. A transcriptomic analyses by RNA-seq identified a large number of genes that are deregulated in response to Pi deficiency that has not been previously identified, likely by the negative influence of light. We are characterizing some of these genes. One of them (PSRR1) seems to be related to S-adenosyl-Methionine (SAM or AdoMet) pathway. By genetic and transcriptomic analyses, we are exploring the novel role of this compound in the Pi response.



Figure 4: Root illumination potentiates Pi starvation toot growth inhibition. A) Phenotype of  12 day old Arabidopsis seedling grown with root in presence of light (LGR- Light Grown Roots) or in darkness (DGR-Dark Grown Roots). B) Root growth quantification in A. (low Pi corresponds to 5 µM of Pi).







In addition, we have found that Pi starvation response in roots is controlled by the balance of different hormones (Figure 5). In this regard, we found that different isomers of cytokinin have specific roles, controlling differentially gene expression, cell division and root growth.



Figure 5: Hormonal treatments alters root/shoot ratio and Pi distribution in dark-grown roots (DGR) in Pi starvation plants.A) Arabidopsis seedlings were grown for 5 days in full medium and then they were transferred to a medium containing IAA, GA, ABA, or Zeatin for 5 more days. B) Weight Root/Shoot ratio from plants treated with different hormones in +Pi or low Pi. C) Pi accumulation in roots or shoots diring plant treatment with different hormones in presence of high phosphate or low Pi. Values are the means of five different experiments ±SD.







Plants modulate molecular responses to adapt development to surrounding environment. The molecular function of cell types during adaptation to Pi starvation is unknown but might involve cellular specialization based on morphological changes. We are analyzing gene expression (transcriptome) and translation (translatome) during Pi starvation response at the cell type level, reconstruct a functional network of cellular adaptation and identify regulators required to sustain plant growth and Pi uptake. This information will be used to generate a transcription/translation map of the Pi starvation response in roots.

Finally, using the D-Root, we are carrying out a screening of Pi accumulation in different Arabidopsis ecotypes. We have identified already one ecotype that seems to accumulate more than 5-fold than Columbia. We are initiating the mapping of this character. Some fungus can colonize roots and transfers phosphorus to its host, increasing plant growth. Using the D-Root system, we will screen for the most efficient combinations of Arabidopsis ecotypes and endophytic fungi, in terms of Pi uptake and growth.

Selected articles: (For a full list of articles visit ORCID web 0000-0002-4113-457X)

González-García, M.-P., Bustillo-Avendaño, E., Sanchez-Corrionero, A., del Pozo, J.C., Moreno-Risueno, M.A. 2020. Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root Cell Populations. Plants 9, 499. DOI: 10.3390/plants9040499

Olmo, R., Cabrera, J., Díaz‐Manzano, F.E., Ruiz‐Ferrer, V., Barcala, M., Ishida, T., García, A., Andrés, M.F., Ruiz‐Lara, S., Verdugo, I., Ochoa, M.P., Fukaki, H., del Pozo, J.C., Moreno‐Risueno, M.Á., Kyndt, T., Gheysen, G., Fenoll, C., Sawa, S., Escobar, C. 2020. Root-knot nematodes induce gall formation by recruiting developmental pathways of post-embryonic organogenesis and regeneration to promote transient pluripotency. New Phytologist. DOI: 10.1111/nph.16521

Conesa, C.M., Saez, A., Navarro-Neila, S., de Lorenzo, L., Hunt, A.G., Sepúlveda, E.B., Baigorri, R., Garcia-Mina, J.M., Zamarreño, A.M., Sacristán, S., del Pozo, J.C. 2020. Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability. Plants 9, 251. DOI: 10.3390/plants9020251

Silva‐Navas, J., Conesa, C.M., Saez, A., Navarro‐Neila, S., Garcia‐Mina, J.M., Zamarreño, A.M., Baigorri, R., Swarup, R., del Pozo, J.C. 2019. Role of cis-zeatin in root responses to phosphate starvation. New Phytologist. DOI: 10.1111/nph.16020

Telléz‐Robledo, B., Manzano, C., Saez, A., Navarro‐Neila, S., Silva‐Navas, J., de Lorenzo, L., González‐García, M.-P., Toribio, R., Hunt, A.G., Baigorri, R., Casimiro, I., Brady, S.M., Castellano, M.M., Del Pozo, J.C. 2019. The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses. The Plant Journal. DOI: 10.1111/tpj.14416

Sayas, E., Pérez‐Benavente, B., Manzano, C., Farràs, R., Alejandro, S., del Pozo, J.C., Ferrando, A., Serrano, R. 2018. Polyamines interfere with protein ubiquitylation and cause depletion of intracellular amino acids: a possible mechanism for cell growth inhibition. FEBS Letters. DOI: 10.1002/1873-3468.13299

Bustillo-Avendaño, E; Ibáñez, S; Sanz, O; Sousa Barros, JA; Gude, I; Perianez-Rodriguez, J; Micol, JL; Del Pozo, JC; Moreno-Risueno, MA; Pérez-Pérez, JM. 2018. "Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis". Plant Physiology. DOI: 10.1104/pp.17.00980".

Manzano, C; Pallero-Baena, M; Silva-Navas, J; Navarro Neila, S; Casimiro, I; Casero, P; Garcia-Mina, JM; Baigorri, R; Rubio, L; Fernandez, JA; Norris, M; Ding, Y; Moreno-Risueno, MA; del Pozo, JC. 2017. "A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development". Journal of Experimental Botany. DOI: 10.1093/jxb/erx324".

Ramirez-Parra, E; Perianez-Rodriguez, J; Navarro-Neila, S; Gude, I; Moreno-Risueno, MA; del Pozo, JC. 2016. "The transcription factor OBP4 controls root growth and promotes callus formation". New Phytologist. DOI: 10.1111/nph.14315".

Fernández-Marcos, M; Desvoyes, B; Manzano, C; Liberman, LM; Benfey, PN; del Pozo, JC; Gutierrez, C. 2016. "Control of Arabidopsis lateral root primordium boundaries by MYB36". New Phytologist. DOI: 10.1111/nph.14304".

Garrido-Arandia, M; Silva-Navas, J; Ramírez-Castillejo, C; Cubells-Baeza, N; Gómez-Casado, C; Barber, D; Pozo, JC; Melendi, PG; Pacios, LF; Díaz-Perales, A. 2016. "Characterisation of a flavonoid ligand of the fungal protein Alt a 1". Scientific Reports. DOI: 10.1038/srep33468".

del Pozo, JC. 2016. "Reactive Oxygen Species: from harmful molecules to fine-tuning regulators of stem cell niche maintenance". PLoS Genetics. DOI: 10.1371/journal.pgen.1006251".

Silva-Navas, J; Moreno-Risueño, MA; Manzano, C; Téllez-Robledo, B; Navarro-Neila, S; Carrasco, V; Pollmann, S; Gallego, FJ; del Pozo, JC. 2016. "Flavonols mediate root phototropism and growth through regulation of proliferation-to-differentiation transition". Plant Cell. DOI: 10.1105/tpc.15.00857".

del Pozo, JC; Ramirez-Parra, E. 2015. "Whole genome duplications in plants: an overview from Arabidopsis". Journal of Experimental Botany. DOI: 10.1093/jxb/erv432".

Silva-Navas, J; Moreno-Risueno, MA; Manzano, C; Pallero-Baena, M; Navarro-Neila, S; Téllez-Robledo, B; Garcia-Mina, JM; Baigorri, R; Javier Gallego, F; del Pozo, JC. 2015. "D-Root: a system to cultivate plants with the root in darkness or under different light conditions". Plant Journal. DOI: 10.1111/tpj.12998".

del Pozo, JC; Ramirez-Parra, E. 2014. "Deciphering the molecular bases for drought tolerance in Arabidopsis autotetraploids". Plant Cell and Environment. DOI: 10.1111/pce.12344".

Lario, LD; Ramirez-Parra, E; Gutierrez, C; Spampinato, CP; Casati, P. 2013. "ANTI-SILENCING FUNCTION1 proteins are involved in ultraviolet-induced DNA damage repair and are cell cycle regulated by E2F transcription factors in Arabidopsis". Plant Physiology. DOI: pp.112.212837 [pii] 10.1104/pp.112.212837".

Bratzel F, Yang C, Angelova A, López-Torrejón G, Koch M, del Pozo JC, Calonje M. (January 2012) Regulation of the new imprinted gene AtBMI1C during development requires the interplay of different epigenetic mechanisms. Molecular Plant. 5: 260-269.

Rodríguez-Herva, J. J., González-Melendi, P., Cuartas-Lanza, R., Antúnez-Lamas, M., Río-Alvarez, I., Li, Z., López-Torrejón, G., Díaz, I., del Pozo, J. C., Chakravarty, S., Collmer, A., Rodríguez-Palenzuela, P. y López-Solanilla, E. 2012. "A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses." Cellular Microbiology. 14(5):669-681.