Plants are sessile organisms that have to adapt their growth to the surrender environment. Therefore, these organisms have adapted a post-embryonic development that allows them to modulate growth depending on internal (genetic) or external signals.

Graphic representation of lateral root formation model. Adapted from Beeckman et al 2001.

Our group is interested in studying the molecular mechanisms that determine the position and the number of lateral roots. For this we are taking genetic approach, looking for mutants that have altered the expression of marker that is positioning where the lateral roots will be developed in the future. In addition to this, we are starting a novel proteomic approach to study the evolution of the root proteome during the process of lateral root primordia formation and development. Ubiquitin (UBQ) is a small peptide that is covalently attached to targets proteins, labelling them for degradation in the 26S-proteasome. The UBQ is activated by the E1 in an ATP-dependent reaction.

The second step is carried out by the E2. The final step, attachment to the targets, involves the activity of the E3 ligases of ubiquitin. Finally, the targets labelled with a poli-UBQ chain are driven to the proteasome, where the deubiquitinases activities removed the poli-UBQ and degraded them. In our laboratory we are studying the role of the ubiquitin pathway in the control of cell division, development and lateral root formation. We are using two different approaches to study this:

• Using specific resins that bind ubiquitinated proteins we are trying to isolate proteins that are modified in vivo with Ub and then identify them by MS.
• Using liquid 2D chromatography analyses of plant treated with or without the proteasome inhibitor MG132. In this case, we are trying to identify proteins that accumulate after a sort treatment with the inhibitor.

Relevant results

During the last years we have initiated the study of the root system development, using Arabidopsis as a model plant. We have identified a gene, called LRIP2, that is expressed in few pericycle cells along the main root, defining the cells that are competent to form LR, in other words, positioned for the formation of the LR (Lateral Root Initiation Points, LRIP; Figure 2). Therefore, we have a perfect genetic marker to study the molecular mechanisms that controls the LR position and number along the main root and we are also in a good position to identify the factors that make one group of pericycle cells competent to form LR versus the adjacent ones that are not competent. Using plants that harbour the promoter LRIP2 gene fused to the reporter GUS gene, we have identified mutants that have reduced or no expression in the LRIP.

Expression pattern of LRIP2 gene

Recently, we have reported that the stability of the transcriptional factor DPB is regulated by the UBQ-pathway, and that specifically the SCFSKP2A but not the SCFSKPB controls such degradation (del Pozo et al., 2006).

Preliminary results have showed that this DPB stability seems to be controlled somehow by auxin, since plants treated with this hormone accumulated more DPB protein. In the near future we want to study in depth the effect of the auxin signalling on the DPB degradation. On the other hand, using the biological tools that we have generated, we pretend to identify the “degron” into the DPB sequence (destruction regulatory motifs) and the sequences of SKP2A that interact with DPB. At present, we are studying the regulation of the SKP2A and SKP2B genes. We have found that SKP2A is regulated at the transcriptional level, but also the SKP2A protein is regulated by degradation through the UB/26S pathway. We have developed a new proteomic approach, using the p62 resin that has high affinity for ubiquitinated proteins, to massively purify proteins regulated by Ub-attachment and subsequent identification by MS. At present, using this approach we have identified more than 200 proteins that are likely to be regulated by ubiquitination. Some of the identified proteins have been described as ubiquitinated in other organisms. Other proteins have been recently reported as UBQ-regulated, such as GIGANTEA or CULLINA 1. Although now we have to validate this approach by testing whether these proteins are ubiquitinated in vivo, the fact the we identified low abundant proteins and that some are indeed regulated by ubiquitination suggest us that our approach can be used for identifying proteins modified with UBQ. During the last years, we have actively collaborated with Dr. C. Gutierrez (CSIC, Madrid) in analyzing the stability of protein involved in DNA replication and the RBR o transcription factors E2F/DP. Likewise, we are collaborating with Dr. M. Estelle (Indiana University), to study the regulation of KRP1 by proteolysis. Our data indicate that SKP2B, but not SKP2A, functions in the degradation of this CDK-inhibitor. Recent data in our lab indicated KRP2, which has been described as a putative target of the UBQ-pathway, interacts with SKP2B. So, we are really interested in studying whether the regulation of KRP2 by the UBQ-dependent degradation and the possible role of the SKP2A or SKP2B in this process. To do this, we have collaboration with Dr. De Veylder (Ghate University, Belgium). We also maintain collaborations with Dr. Solano at the CNB, Dr. Micol (UMH) and Dr Casero at the University of Extremadura.

Recent Publications

Bratzel, F.; Lopez-Torrejon, G.; Koch, M.; del Pozo, J.C.; Calonje, M. 2010. "Keeping Cell Identity in Arabidopsis Requires PRC1 RING-Finger Homologs that Catalyze H2A Monoubiquitination". Current Biology. 20(20):1853-1859

Desvoyes, B.; Sanchez, M.P.; Ramirez-Parra, E.; Gutierrez, C. 2010. "Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development". Heredity. 105(1):80-91

Perez-Perez, J.M.; Candela, H.; Robles, P.; Lopez-Torrejon, G.; del Pozo, J.C.; Micol, J.L. 2010. "A Role for AUXIN RESISTANT3 in the Coordination of Leaf Growth". Plant and Cell Physiology. 51(10):1661-1673

Jurado, S.; Diaz-Trivino, S.; Abraham, Z.; Manzano, C.; Gutierrez, C.; del Pozo, J.C. 2008. "SKP2A, an F-box protein that regulates cell division, is degraded via the ubiquitin pathway". Plant Journal. 53(5):828-841

Jurado, S.; Trivino, S.D.; Abraham, Z.; Manzano, C.; Gutierrez, C.; del Pozo, J.C. 2008. "SKP2A protein, an F-box that regulates cell division, is degraded via the ubiquitin pathway.". Plant Signal Behav. 3(10):810-2

Manzano, C.; Abraham, Z.; Lopez-Torrejon, G.; del Pozo, J.C. 2008. "Identification of ubiquitinated proteins in Arabidopsis". Plant Molecular Biology. 68(1-2):145-158

Ren, H.; Santner, A.; del Pozo, J.C.; Murray, J.A.H.; Estelle, M. 2008. "Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases". Plant Journal. 53(5):705-716

Barrero, J.M.; Gonzalez-Bayon, R.; del Pozo, J.C.; Ponce, M.R.; Micol, J.L. 2007. "INCURVATA2 encodes the catalytic subunit of DNA polymerase alpha and interacts with genes involved in chromatin-mediated cellular memory in Arabidopsis thaliana". Plant Cell. 19:2822-2838

del Pozo, J.C.; Diaz-Trivino, S.; Cisneros, N.; Gutierrez, C. 2007. "The E2FC-DPB Transcription Factor Controls Cell Division, Endoreplication and Lateral Root Formation in a SCF-Dependent Manner". Plant signaling & behavior. 2(4):273-274

Ramirez-Parra, E.; del Pozo, J.C.; Desvoyes, B.; Sanchez, M.P.; Gutierrez, C. “E2F-DP transcription factors” In: Cell Cycle Control and Plant Development. D. Inzé, Ed.; pp. 138-162, Blackwell Publishing, Oxford (2007); Annu. Plant. Rev. 32; ISBN: 978-1-4051-5043-9