Factores transcripcionales de la clase DOF en la regulación génica en semillas

Los factores transcripcionales de la clase DOF son una familia de proteínas implicada en la regulación de la expresión génica de procesos específicos de plantas que se unen a la secuencia 5'-(T/A)AAAG-3' en los promotores de los genes que regulan. La presente tesis versa sobre: 1,- La identificación y anotación de nuevos genes DOF de cebada. 2,- La caracterización molecular y funcional de dos nuevos factores DOF de esta especie implicadas en la regulación de genes específicos de semillas. 3,- La realización de un análisis filogenético evolutivo de la familia DOF en siete especies representativas de la evolución de las plantas, el alga verde Chlamydomonas reinhardtii, el musgo Physcomitrella patens, el helecho Selaginella moellendorffii, la gimnosperma Pinus taeda, la angiosperma dicotiledónea Arabidopsis thaliana y las dos angiospermas moncotiledóneas Oryza sativa y Hordeum vulgare. En C.reinhardtii hemos identificado un único miembro de la familia DOF, en P.patens nueve, y ocho en S.moellendorffti y P.taeda. En cebada, nuestra especie de interés, se han identificado y anotado 18 nuevos miembros de esta familia en las bases de datos, que se suman a los ocho previamente anotados en este laboratorio. Para profundizar en el origen y evolución de esta familia de factores transcipcionales, realizamos un análisis filogenético con los DOFs previamente identificados y aquellos de A.thaliana y O.sativa. Todos estos genes pueden agruparse en seis familias de genes ortólogos y parálogos que se habrían originado desde un grado basal parafilético o subfamilia A. La estructura de intrones y exones del gen DOF de C.reinhardtii apoya la formación del primer DOF por barajado de exones en el ancestro de las plantas. Así, duplicaciones del DOF ancestral durante el subsiguiente proceso evolutivo habría ocasionado la formación de una familia de noventa y dos miembros en las plantas vasculares aquí estudiadas. Nuestros datos, sugieren además que la pérdida, adquisición y barajado de motivos conservados además del DOF entre los nuevos genes, habría condicionado la formación de las siete subfamilias aquí descritas. Entre los ventiséis genes DOF anotados en cebada, HvDof17 y HvDof19 fueron seleccionados para su caracterización posterior, ya que se expresaban preferentemente en la aleurona de las semillas en germinación. En la aleurona post-germinativa de los granos de creales, se sintetizan un conjunto de hidrolasas, cuya expresión está inducida por GA y reprimida por ABA, que posteriormente se secretan en el endospermo amiláceo liberando los nutrientes que se movilizaran hacia el embrión. En los promotores de estos genes se ha identificado un motivo tripartito en cis necesario para una respuesta plena a GA (GARC: GA-Responsive Complex). Por el contrario, los elementos regulatorios en cis que median la respuesta de estos genes al ABA son desconocidos. Los tránscritos del gen HvDof19 se acumulan en la capa de aleurona en respuesta a ABA, estando su expresión, así como la del otro gen seleccionado, HvDof17, reprimida por GA. En este tejido, HvDOF19 media la represión por ABA del gen CatB, que codifica una tiol proteasa del tipo catepsina B, y HvDIF17 contribuye a la regulación negativa de este gen en una ruta independiente de esta hormona. Ambos factores son capaces de revertir la trans-activación del gen CatB medida por GAMYB, el cual pertenece a la clase de factores R2R3MYB y está implicado en la activación de la expresión de genes de hidrolasas en respuesta a GA; e interaccionan con él in vivo. En el endospermo en desarrollo de cebada, hemos determinado que HvDOF 17 y HVDOF19 también regulan la expresión del gen Hor2, que codifica la proteína de reserva B-hordeína. El patrón de expresión de HvDof19 durante la maduración del endospermo, se ajusta a su comportamiento como activador transcripcional del gen Hor2 y a la interacción detectada entre éste y BLZ2, un activador de la expresión de este mismo gen previamente caracterizado en el laboratorio. Por el contrario, HvDIF 19 reprime la expresión del gen Hor2. En conclusión, esta tesis contribuye a un mejor conocimiento de la regulación transcripcional de los genes de hidrolasas en respuesta a ABA, y muestra la implicación de HvDOF19 y HvDOF17 en la activación y represión, respectivamente, de los genes que codifican proteínas de reserva en la semilla de cebada

Post-embryonic organogenesis and plant regeneration from tissues: two sides of the same coin?

Plants have extraordinary developmental plasticity as they continuously form organs during post-embryonic development. In addition they may regenerate organs upon in vitro hormonal induction. Advances in the field of plant regeneration show that the first steps of de novo organogenesis through in vitro culture in hormone containing media (via formation of a proliferating mass of cells or callus) require root post-embryonic developmental programs as well as regulators of auxin and cytokinin signaling pathways. We review how hormonal regulation is delivered during lateral root initiation and callus formation. Implications in reprograming, cell fate and pluripotency acquisition are discussed. Finally, we analyze the function of cell cycle regulators and connections with epigenetic regulation. Future work dissecting plant organogenesis driven by both endogenous and exogenous cues (upon hormonal induction) may reveal new paradigms of common regulation

FUSCA3 from barley unveils a common transcriptional regulation of seed-specific genes between cereals and Arabidopsis.

Accumulation of storage compounds in the embryo and endosperm of developing seeds is a highly regulated process that allows seedling growth upon germination until photosynthetic capacity is acquired. A critical regulatory element in the promoters of seed storage protein (SSP) genes from dicotyledonous species is the RY box, a target of B3-type transcription factors. However, the functionality of this motif in the transcriptional regulation of SSP genes from cereals has not been fully established. We report here the identification and molecular characterization of barley FUSCA3, a B3-type transcription factor as yet uncharacterized in monocotyledonous plants. Our results show that both the barley and Arabidopsis FUS3 genes maintain a conserved functionality for the regulation of SSP genes and anthocyanin biosynthesis in these two distantly related phylogenetic groups. Complementation of the loss-of-function mutant fus3 in Arabidopsis by the barley HvFus3 gene resulted in restored transcription from the At2S3 gene promoter and normal accumulation of anthocyanins in the seed. In barley, HvFUS3 participates in transcriptional activation of the endosperm-specific genes Hor2 and Itr1. HvFUS3, which specifically binds to RY boxes in EMSA experiments, trans-activates Hor2 and Itr1 promoters containing intact RY boxes in transient expression assays in developing endosperms. Mutations in the RY boxes abolished the HvFUS3-mediated trans-activation. HvFus3 transcripts accumulate in the endosperm and in the embryo of developing seeds, peaking at mid maturation phase. Remarkably, HvFUS3 interacts with the Opaque2-like bZIP factor BLZ2 in yeast, and this interaction is essential for full trans-activation of the seed-specific genes in plant

Flavonols Mediate Root Phototropism and Growth through Regulation of Proliferation-to-Differentiation Transition

Roots normally grow in darkness, but they may be exposed to light. After perceiving light, roots bend to escape from light (root light avoidance) and reduce their growth. How root light avoidance responses are regulated is not well understood. Here, we show that illumination induces the accumulation of flavonols in Arabidopsis thaliana roots. During root illumination, flavonols rapidly accumulate at the side closer to light in the transition zone. This accumulation promotes asymmetrical cell elongation and causes differential growth between the two sides, leading to root bending. Furthermore, roots illuminated for a long period of time accumulate high levels of flavonols. This high flavonol content decreases both auxin signaling and PLETHORA gradient as well as superoxide radical content, resulting in reduction of cell proliferation. In addition, cytokinin and hydrogen peroxide, which promote root differentiation, induce flavonol accumulation in the root transition zone. As an outcome of prolonged light exposure and flavonol accumulation, root growth is reduced and a different root developmental zonation is established. Finally, we observed that these differentiation-related pathways are required for root light avoidance. We propose that flavonols function as positional signals, integrating hormonal and reactive oxygen species pathways to regulate root growth direction and rate in response to light

Divergent regulation of auxin responsive genes in root-knot and cyst nematodes feeding sites formed in Arabidopsis

13 Pág.Cysts (CNs) and root-knot nematodes (RKNs) induce specialized feeding cells, syncytia, and giant cells (GCs), respectively, within plant roots. The plant tissues around the GCs usually by respond forming a root swelling called a gall that contains the GCs. The ontogenesis of feeding cells is different. GC formation is a process of new organogenesis from vascular cells, which are still not well characterized, that differentiate into GCs. In contrast, syncytia formation involves the fusion of adjacent cells that have already differentiated. Nonetheless, both feeding sites show an auxin maximum pertinent to feeding site formation. However, data on the molecular divergences and similarities between the formation of both feeding sites regarding auxin-responsive genes are still scarce. We studied genes from the auxin transduction pathways that are crucial during gall and lateral root (LR) development in the CN interaction by using promoter-reporter (GUS/LUC)transgenic lines, as well as loss of function lines of Arabidopsis. The promoters pGATA23 and several deletions of pmiR390a were active in syncytia, as were in galls, but pAHP6 or putative up-stream regulators as ARF5/7/19 were not active in syncytia. Additionally, none of these genes seemed to play a key role during cyst nematode establishment in Arabidopsis, as the infection rates in loss of function lines did not show significant differences compared to control Col-0 plants. Furthermore, the presence of only canonical AuxRe elements in their proximal promoter regions is highly correlated with their activation in galls/GCs (AHP6, LBD16), but those promoters active in syncytia (miR390, GATA23) carry AuxRe overlapping core cis-elements for other transcription factor families (i.e., bHLH, bZIP). Strikingly, in silico transcriptomic analysis showed very few genes upregulated by auxins common to those induced in GCs and syncytia, despite the high number of upregulated IAA responsive genes in syncytia and galls. The complex regulation of auxin transduction pathways, where different members of the auxin response factor (ARF) family may interact with other factors, and the differences in auxin sensitivity, as indicated by the lower induction of the DR5 sensor in syncytia than galls, among other factors, may explain the divergent regulation of auxin responsive genes in the two types of nematode feeding sites.This work was supported by the Spanish Government (PID2019-105924RB-I00, Ministerio de Ciencia e Innovacion, MCIN/AEI/10.13039/501100011033 and RED2018-102407-T), by the Castilla-La Mancha Government (SBPLY/17/180501/000287; SBPLY/21/180501/000033), o CE; and by MCIN of Spain and ERDF (grant PID2019-111523GB-I00) to MM-R.Peer reviewe

Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division

11 Pág.Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.This work was supported by the NSF Graduate Research Fellowship Program (DGE-1252376, to N.M.C. and A.P.F.). Research in the R. Simon lab was funded by the Deutsche Forschungsge-meinschaft (Si947/10 and an Alexander von Humboldt Foundation fellowship, to B.B.). This work was also supported by a grant from the Ministerio de Economía y Competitividad of Spain and European Regional Development Fund (BFU2016-80315-P, to M.A.M.-R.). E.B.A. is supported by Ayudante de Investigacion contract PEJ-2017-AI/BIO-7360 from Comunidad Madrid. S.G.Z. was supported by the HHMI and by a grant from the NIH (GM118036). Research in the K.L.G. lab was funded by NSF Grant 1243945. The R. Sozzani lab is supported by an NSF CAREER grant (MCB-1453130) and the North Carolina Agricultural & Life Sciences Research Foundation at North Carolina State University’s College of Agricultural and Life Sciences.Peer reviewe

Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems

Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs.Los sistemas automáticos utilizados en el estudio de la sensibilidad a los antimicrobianos están introducidos en la mayoría de los laboratorios de Microbiología Clínica en España, utilizando principalmente los puntos de corte del European Committee on Antimicrobial Susceptibility Testing (EUCAST). En 2007, un grupo de expertos publicó unas recomendaciones para incluir antimicrobianos y seleccionar concentraciones en estos sistemas. Bajo el auspicio del Comité Español del Antibiograma (COESANT) y del Grupo de Estudio de los Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) y alineado con el Plan Nacional frente a la Resistencia a los Antibióticos (PRAN), un grupo de expertos ha actualizado dicho documento. Las principales modificaciones realizadas sobre la versión anterior comprenden la inclusión de nuevos agentes antimicrobianos, la adaptación de los rangos de concentraciones para cubrir los puntos de corte clínicos y los puntos de corte epidemiológicos (ECOFF) definidos por el EUCAST, y para la inferencia de nuevos mecanismos de resistencia. Esta propuesta debería ser considerada por los diferentes fabricantes y los usuarios cuando se diseñen nuevos paneles o tarjetas. Además, se incluyen recomendaciones para realizar informes selectivos. Con este enfoque, la implementación de los puntos de corte del EUCAST será más fácil, aumentando la calidad de los datos del antibiograma y su interpretación microbiológica. También será de utilidad para los estudios de vigilancia epidemiológica, así como para el uso clínico de los antimicrobianos, de acuerdo con los programas de optimización de uso de antimicrobianos (PROA)

Regulation of organogenesis and tissue patterning during plant postembryonic development

Trabajo presentado al Seminario del CRAG, celebrado el 17 de enero de 2020.Peer reviewe

Characterization of novel developmental cues in Arabidopsis roots

The root system and its functionality is essential for survival of terrestrial plants. Lateral root (LR) formation is a plastic process regulating root system architecture, although locations where LRs may form are pre-patterned by the Lateral Root Clock. Under standard growth conditions, LRs form as repeated units along the root primary axis1,2. The Lateral Root Clock was first defined using the synthetic auxin-response promoter DR5, which pulses rhythmically about every 5 hours in a zone close from the root tip: the Oscillation Zone (OZ). Further analyses of this region showed changes in the expression of thousands of genes that fluctuated in or out of phase with DR53. As cells exit the OZ, oscillations cease and maxima in auxin response (peak of oscillations) are followed by permanent expression of DR5 in specific sites, called Pre-Branch Sites (PBS). At these sites, LR initiation will eventually occur3,4. The OZ arises as a key developmental zone for LR pre-patterning. It is unknown how this zone is positioned and delimited. arf7arf19 mutants do not form LRs, however present constitutive non-oscillating expression of DR5 in the OZ3. Thus, DR5 can be used in arf7arf19 mutant to define the OZ. A genetic screen using Ethyl Methanesulfonate (EMS) was performed in arf7arf19 mutants carrying the DR5:Luciferase marker. Mutagenized F2 populations were searched for alterations in DR5 expression in the OZ. We aimed to find mutants with expanded marker expression, as expansion of the OZ would indicate the existence of a repressing signal, likely unknown, with an antagonistic role to auxin. Here we show the different phenotypes of mutants we have found. These were classified in three groups: A (expanded OZ), B (higher DR5 intensity in the OZ) and C (subtle changes in DR5 but increased LR formation). In addition, we crossed these mutants back to the wild type to remove arf7 and arf19 along with other undesired mutations. Segregating populations were analyzed to determine the effect of mutations in OZ positioning and PBS formation, which are indicative of their putative role as developmental cues. Based on these data, we will prioritize mutants and proceed to map the

Identification of polarization cues downstream of POTENT involved in asymmetric cell division of lateral root founder cells

Plants show a postembryonic mode of development with most organs being made after embryogenesis. Root system establishment requires formation of lateral roots (LR), being the hormone auxin a key player promoting their formation. LR formation involves positioning of cells competent (prebranch sites) to form LR through oscillatory gene activiy1. Subsequently, cells within prebranch-sites are reprogrammed to become LR founder cells (FC). LR initiation starts with asymmetric cell division (ACD) of FCs to eventually give rise to the different cell lineages that make up the root23. ACD is therefore crucial to LR formation and involves polarization of FCs resulting in nuclear migration. How polarization of FCs is established is unknown. Nuclear migration involves auxin signaling and some downstream factors have been identified3. Auxin signaling involves the ubiquitin-mediated degradation of the AUXIN/INDOLE-3- ACETIC ACID (Aux/IAA) transcriptional co-regulators via proteasome3. We have identified a mutant defective in lateral root formation called potent. Potent mutation prevents degradation of an Aux/IAA factor by auxin resulting in inhibition of auxin signaling. As a consequence, over-specification of prebranch-sites and FCs along the root occur. In addition, FCs in potent divide symmetrically generating daughter cells of similar sizes and incorrect cells fates. Furthermore, these daughter cells remain blocked in development and cannot form LRs. We aim to identify polarization cues downstream of POTENT involved in ACD of FCs. For this purpose, we have generated a potent inducible line -by estradiol- and introgressed the FC marker SKP2B to monitor specification and ACD division. Our results show that potent factor dose correlates with FC specification and number of LRs. Higher assessed doses of estradiol caused all pericycle to become FC and preventing LR formation. Upon auxin treatment, FCs remained blocked in development although symmetric divisions were observed indicating that POTENT must control most polarization cues required for ACD. We have performed protoplasting followed by Fluorescent Activated Cell Sorting (FACS) and isolated FCs in the potent inducible line. We will perform RNA sequencing of these cells to identify polarization cues regulated by POTENT