PROJECTS

The main axis of our research career has been the study of the molecular mechanisms that allow plants to integrate environmental and stress signals to translate them into adaptive and developmental responses.

In this sense, we have been fascinated by the complexity of the signaling routes in plants; not only for the regulatory elements that compose them but also for the networks that interconnect them. In fact,studying plant signaling routes is the common theme of all research projects is what we have been involved in including phosphate fast signaling, plant responses to light,flowering time control and circadian clock, and, more recently, regulation of abscissaic acid signaling (ABA). The fact that all these processes are regulated by the ubiquitin-proteasome system, which controls the stability and, therefore, the activity of hundreds, if not thousands, of proteins in plants, and where E3 ubiquitin ligase enzymes can recognize different target proteins raises several key questions that we intend to answer:

- How is the modulation of different signaling pathways and the corresponding adaptive responses of the plant coordinated at the ubiquitin-proteasome system level?

- Is there a hierarchy between different signaling routes regulated by the same components of the ubiquitin-proteasome system? - How is the activity of the ubiquitin-proteasome system itself modulated in response to environmental and intracellular signals?

To answer these questions we have chosen Cullin 4 RING ligases (CRL4) for our studies as a representative group of E3 ubiquitin ligases, since they coordinate different biological processes in plants in a coordinated way, including photomorphogenesis, ABA signaling, repair of damaged DNA, circadian clock function, flowering, etc ...(figure 1 ).To date, our studies have revealed new functions for CRL4 and a new regulatory mechanism, mediated by ABA, that controls their activity, in addition to direct connections with events Chromatin remodelers, which we describe in more detail later.

Figure 1: CRL4 E3 ubiquitin ligases regulate multiple processes, which span the plant ́s whole life, including embryogenesis, seed germination, seedling photomorphogenesis, circadian clock function and flowering. As well, CRL4s control different abiotic stress responses such as drought tolerance, adaptation to cold stress, nutrient deprivation and DNA damage responses.

In another section, although closely related to the previous one, we are very interested in the applied aspects of our research. Thus, aware that the manipulation of the genes we work with in species of agronomic interest can generate interesting and beneficial traits for breeders and farmers, our group has started a line of research dedicated to obtaining stress-tolerant crops. We consider that the results obtained by our group to date have only managed to reveal a minimal portion of the regulatory potential of E3 ubiquitin ligases to interconnect different responses to stress and development. During the next years our main objective is to increase our knowledge about this integrating mechanism. Therefore, we propose:

-Characterize the molecular mechanisms by which ABA controls the activity of CRL4-DDA1 complexes. Our data shows that ABA promotes the disassembly of these complexes, leading to their inactivation. Such a mechanism of inhibition of a hormone mediated E3 ubiquitin ligase has not been previously described in any biological system. Our results show that this process does not require de novo protein synthesis since all the necessary factors are present in the cell when ABA signaling is triggered. Our objective is to identify these factors and characterize their inactivating function of CRL4-DDA1 complexes.

-Determine how a specific E3 ubiquitin ligase allows the interrelation between different signal transduction pathways. In this sense, through the purification of plant protein complexes using TAP techniques (Tandem Affinity Purification), we have observed that substrate adapters involved in the control of photomorphogenesis and ABA signaling can coexist in the same complexes. E3 ubiquitin ligase CRL4, thus generating a platform for the interrelation of stress and developmental signals. It should be noted that we have found that these complexes can be associated with chromatin and epigenetic modification machinery (specifically with the SAGA complex involved in histone acetylation), which could allow the almost direct conversion of these signals in changes in gene expression.

-Characterize the mechanisms by which the ALIX protein regulates the traffic of different cargo proteins in plant cells. As indicated above, ALIX participates in the trafficking of various membrane-associated proteins, although using different endosomal compartments for this. In this context, ALIX acts as a ubiquitin receptor. In this way, ALIX recognizes and classifies cargo proteins by binding to them once they have been conjugated with ubiquitin. However, our data shows direct interaction of ALIX with specific target proteins, including the ABA receptors of the PYR / PYL family. We aim to characterize the molecular bases that confer target specificity to ALIX and to study how ALIX function contributes to growth and adaptation to water stress (mediated by ABA) in plants. -To delve into the applied aspects of our research. Currently, we continue to generate tools that allow us to manipulate the expression and activity of E3 ubiquitin ligase CRL4 in crops of agronomic interest in order to increase their tolerance to water stress