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

An auxin-regulable oscillatory circuit drives the root clock in Arabidopsis

CSIC - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.This work was funded by the Ministerio de Economía y Competitividad of Spain (MINECO) and/or the ERDF (BFU2016-80315-P to M.A.M.-R., BIO2017-82209-R to J.C.d.P., and TIN2016-81079-R to A.R.-P.), the Comunidad de Madrid and/or ERDF and ESF (2017-T1/BIO-5654 to K.W. and S2017/BMD-3691 to A.R.-P.), the Howard Hughes Medical Institute and the NIH (R35-GM131725 to P.N.B.), the Fonds Wetenschappelijk Onderzoek (FWO Flanders) (G022516N, G020918N, and G024118N to T.B.), and the “Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigacion of Spain [SEV-2016-0672 (2017–2021)] to K.W., P.P.-G., and M.A.M.-R. through CBGP. M.M. was supported by a postdoctoral contract associated to SEV-2016-0672, E.B.-A. by Ayudante de Investigacion contract PEJ-2017-AI/BIO-7360 from the Comunidad de Madrid, A.S.-C. and L.S.-R. by FPI contracts from MINECO (BES-2014-068852 and BES-2017-080155, respectively), J.C. by a Juan de la Cierva contract from MINECO (FJCI-2016-28607), P.P.-G. by a Juan de la Cierva contract from MINECO (FJCI-2015-24905) and Programa Atraccion Talento from Comunidad Madrid (2017-T2/BIO-3453), A.S. by a Torres Quevedo contract from MINECO (PTQ-15-07915), and K.W. by program PGC2018-093387-A-I00 from the Ministerio de Ciencia e Innovacion (MICIU)Peer reviewe

Identificación y perturbación de la señalización auxínica por el factor Aux/IAA POTENT en el Reloj de la Raíz y la formación de raíces laterales

Las plantas presentan una gran plasticidad para modular su arquitectura radicular en función de las condiciones endógenas y ambientales en las que se encuentra. Los sitios competentes o sitios de pre-ramificación (PBS) a partir de las cuales se forman las raíces laterales (RL) está determinado por un mecanismo oscilatorio que resulta de la actividad del Reloj de la Raíz en la Zona de Oscilación (ZO). El Reloj de la Raíz es, por lo tanto, el mecanismo que inicia la formación de RL y el que permite a la planta modular su arquitectura. En él se establecen ciclos de respuesta auxínica y otras respuestas aun por caracterizar coincidentes con la expresión periódica de oscilaciones génicas, que culminan en la generación de PBS cíclicamente. Dentro de la maquinaria oscilatoria del Reloj se conocen dos módulos de señalización, IAA28 que controla la respuesta necesaria para mantener la amplitud de las oscilaciones e IAA18/POTENT-AUXIN RESPONSE FACTOR (ARF) 7, que regula la periodicidad. Sin embargo, se desconocen muchos aspectos de la regulación e integración la maquinaria oscilatoria Reloj de la Raíz y como las alteraciones de esta maquinaria a su vez repercuten en la regulación de la arquitectura radicular. Secuencialmente, los procesos de iniciación de RL requieren de una especificación a células fundadoras de raíz lateral (CFRL) partir de los PBS. Este proceso de iniciación requiere de la división formativa anticlinal de las CFRL generando como resultado células de distinto tamaño y cambios en el destino celular, proceso que también requiere de la señalización por auxinas. A su vez, en los procesos de iniciación de la organogénesis de raíces laterales se están descubriendo vías de regulación mediadas por péptidos que influyen en las divisiones asimétricas. Cabe destacar que los mecanismos de polarización asociados a las CFRL no se conocen con exactitud. Sin embargo, la ganancia de función de IAA18/POTENT (mutante potent) impide la señalización necesaria para que establecer la asimetría y/o polarización de las CFRL, y por lo tanto estas se dividen simétricamente. Por todo ello, las líneas de investigación de la presente tesis han sido: profundizar en el impacto del módulo de señalización IAA18/POTENT sobre distintos componentes del oscilador del Reloj de la Raíz y en la polarización de CFRL, así como en la identificación de reguladores dicha polarización a partir del análisis transcriptómico de CFRL tras la inducción de potent. En cuanto los resultados obtenidos con respecto al funcionamiento del Reloj de la Raíz, hemos determinado que IAA18/POTENT a través de su represión sobre la actividad de ARF7 sería capaz de regular positivamente los niveles de auxinas en la ZO, presumiblemente por la inducción de genes implicados en la biosíntesis de auxinas. Basándonos en papel de ARF7 en la regulación del Reloj de la Raíz en la ZO y en papel redundante que presenta con su homólogo más cercano ARF19, hemos analizado el papel de ARF19 en la ZO y hemos determinado a través del uso del reportero DR5:Luciferasa y de los mutantes arf19-1 y arf7-1 arf19-1 el papel de ARF19 como represor de las oscilaciones, , y en la regulación de la adquisición de competencia para formar PBS o priming, que es además redundante con la función de ARF7. Además, gracias al análisis de DR5:Luciferasa junto al tratamiento de auxinas y análisis temporal de los PBS en el mutante arf7-1 hemos detectado que ARF7 presenta un rol bimodal y que aparte de presentar una actividad represora ya conocida en la ZO, tiene una función activadora manteniendo los PBS y la señalización auxina, lo que permite la progresión en el proceso de organogénesis de RL. Para intentar entender la regulación autónoma de las oscilaciones, nos hemos basado en simulaciones in silico que predicen la regulación de las oscilaciones en antifase de ARF7, por un elemento oscilante en fase o mediante la modulación del heterodímero entre IAA18/POTENT y ARF7 por factor desconocido que responda a auxinas. A través de tratamientos con auxinas y el análisis de genes en fase cuyos mutantes muestran formación comprometida de PBS, hemos determinado que las auxinas exógenas y al menos los genes en fase SHATTERPROOF no son capaces de modular la expresión ni los niveles de ARF7. Sin embargo, hemos determinado que la dosis de potent es capaz de modular negativamente los niveles de expresión de ARF7 en la OZ planteando una vía autónoma en la regulación de las oscilaciones. A través de la modulación de los niveles de potent mediante el uso de una línea inducible por estradiol, hemos determinado una alta sensibilidad del Reloj a concentraciones bajas de potent, observando una correlación positiva entre los niveles de potent y número de sitios de pre-ramificación, espaciamiento de los mismos y número de células fundadoras, y además en el número de raíces laterales cuando se realizaron tratamiento conjuntos con auxinas. Asimismo, la inducción de potent, resulta en la activación de genes en fase como LOB-DOMAIN (LBD) 16, lo que podría implicar la inhibición tanto de la actividad represora de ARF7 en la ZO como la de su función activadora en la Zona de diferenciación (ZD). Nuestros resultados a través de microscopía laser confocal muestran que la CFRL en potent se dividen únicamente de forma simétrica siendo incapaces de generar divisiones formativas asimétricas, las cuales son imprescindibles para el desarrollo de la RL. Para entender la cascada de señalización mediada por IAA18/POTENT en la regulación de la polarización de las CFRL y subsiguiente división asimétrica analizamos transcripcionalmente mediante ARNseq las CFRL tras la inducción de potent y en tratamiento conjuntos con auxinas.. En este proceso, hemos optimizado los protocolos de extracción de protoplastos de tejidos poco abundantes e internos como lo son las CFRL en el periciclo. El análisis del ARNseq de las CFRL tras la inducción de potent y tras la inducción de potent y el tratamiento con auxinas, muestra un total de 3811 genes diferencialmente expresados con potencial para controlar algún aspecto de la polarización de las CFRL y primera división asimétrica. Dado el papel de IAA18/POTENT en la señalización auxínica analizamos su regulación sobre los factores de transcripción LBDs que tienen un papel en las divisiones asimétricas de la CFRL. La mayoría de los LBDs diferencialmente expresados se encuentran reprimidos indicando la activación de rutas de respuesta a auxinas previamente descritas junto con otras aún desconocidas. La señalización por los péptidos GLV6/GLV10 y TOLS2/PIP2 juega un papel importante inhibitorio en la regulación de las divisiones anticlinales del periciclo y extrapolable a CFRL a través de la fosforilación de la MPK6 por receptores quinasa que a su vez implicaría aguas abajo la actividad de los factores WRKY23, PLT5 y PUCHI entre otros. A través de la comparativa entre los nuestros datos de RNAseq y aquellos previamente descritos para GLV6 y TOLS2, hemos determinado que apenas existe solapamiento entre ambas cascadas de señalización. Asimismo, mediante ensayos de inducción de potent en el mutante mpk6, hemos determinado que la regulación de la polarización y divisiones asimétricas de CFRL mediada por IAA18/POTENT no requiere de la actividad de la MPK6, lo que confirma que dicha regulación es independiente de GLV6/GLV10 y TOLS2/PIP2. Con el objetivo de cribar los genes diferencialmente expresados que fueran candidatos a regular la polarización y divisiones asimétricas de las CFRL utilizamos el LRMap previamente generado en nuestro laboratorio y que contiene los niveles de expresión de todos los genes a lo largo de una cronología del desarrollo de raíces laterales en los estadios tempranos. En dicho análisis nos centramos en el estadio I y especialmente en el comportamiento de los genes entre CFRL antes y después de la migración nuclear (primera indicación morfológica de la polarización de las CFRL). A través del análisis fenotípico de mutantes hemos identificado la proteína KINASE FAMILY PROTEIN (KFP) 1 como necesaria para la polarización y divisiones asimétricas de las CFRL aguas abajo de la cascada de señalización IAA18/POTENT. Dado que las CFRL en kfp1 se tienden a dividir simétricamente, procedimos a comprobar la localización de la proteína KFP1 y determinar si existía expresión en las CFRL. KFP1 se encuentra en la mayoría de las membranas celulares, incluyendo las CFRL, y no localizándose en la membrana de las células formadas por divisiones anticlinales posteriores a la primera división asimétrica, ni en las divisiones periclinales que dan lugar al estadio II indicando una especificidad por su regulación en la polarización de la primera división asimétrica. Al tratarse de un gen aguas abajo de auxinas nos preguntábamos si la proteína KFP1 respondería a la adición de auxinas, Los niveles de la proteína KFP1, según se esperaba responden positivamente a la adición exógena de auxinas CFRL. Finalmente, hemos determinado que la falta de asimetría en las CFRL del mutante kfp1 se asocia con la ausencia de localización asimétrica del proceso de reciclaje de membranas por endocitosis así como con la deslocalización de marcadores polarizados durante la migración nuclear como MAKR4 y PIN1. ----------ABSTRACT---------- Plants have great plasticity to modulate their root architecture depending on the endogenous and environmental conditions in which they are found. The competent sites or pre-branching sites (PBS) from which the lateral roots (RL) are formed is determined by an oscillatory mechanism that results from the activity of the Root Clock in the Oscillation Zone (ZO). The Root Clock is therefore the mechanism that initiates the formation of RL and the one that allows the plant to modulate its architecture. In the Root Clock, auxinic response cycles and other responses yet to be characterized are established, coinciding with the periodic expression of gene oscillations, which culminate in the generation of PBS cyclically. Within the Clock's oscillatory machinery, two signaling modules are known, IAA28, which controls the response necessary to maintain the amplitude of the oscillations, and IAA18/POTENT-AUXIN RESPONSE FACTOR (ARF) 7, which regulates the periodicity. However, many aspects of the regulation and integration of the Root Clock oscillatory machinery and how alterations in this machinery in turn affect the regulation of root architecture are unknown. Sequentially, RL initiation processes require specification of lateral root founder cells (RLCF) from PBS. This initiation process requires the anticlinal formative division of CFRLs, generating as a result cells of different sizes and changes in cell fate, a process that also requires auxin signaling. In turn, regulatory pathways mediated by peptides that influence asymmetric divisions are being discovered in the initiation processes of lateral root organogenesis. It should be noted that the polarization mechanisms associated with CFRL are not exactly known. However, the gain of function of IAA18/POTENT (potent mutant) prevents the necessary signaling to establish the asymmetry and/or polarization of the CFRLs, and therefore they divide symmetrically. For all these reasons, the research lines of this thesis have been: to delve into the impact of the IAA18/POTENT signaling module on different components of the Root Clock oscillator and CFRL polarization, as well as on the identification of regulators such polarization from CFRL transcriptomic analysis after potent induction. Based on the results obtained regarding the functioning of the Root Clock, we have determined that IAA18/POTENT, through its repression over ARF7 activity, would be capable of positively regulating auxin levels in the ZO, presumably by inducing genes involved in auxin biosynthesis. Based on the role of ARF7 in the regulation of the Root Clock in the ZO and its redundant role with its closest homologue ARF19, we have analyzed the role of ARF19 in the ZO and determined through the use of the DR5:Luciferase reporter and of the mutants arf19-1 and arf7-1 arf19-1 the role of ARF19 as a repressor of oscillations, and in the regulation of the acquisition of competence to form PBS or priming, which is also redundant with the function of ARF7. In addition, thanks to the analysis of DR5:Luciferase together with the auxin treatment and temporal analysis of the PBS in the arf7-1 mutant, we have detected that ARF7 has a bimodal role and that apart from presenting a repressive activity already known in the ZO, it has a activating function by maintaining PBS and auxin signaling, which allows progression in the organogenesis process of RL. In order to understand the autonomous regulation of the oscillations, we have based ourselves on in silico simulations that predict the regulation of the anti-phase oscillations of ARF7, by an in-phase oscillating element or by modulation of the heterodimer between IAA18/POTENT and ARF7 by an unknown factor. responsive to auxin. Through auxin treatments and in-phase gene analysis whose mutants show compromised PBS formation, we have determined that exogenous auxins and at least SHATTERPROOF in-phase genes are unable to modulate ARF7 expression and levels. However, we have determined that the dose of potent is capable of negatively modulating the expression levels of ARF7 in the OZ, positing an autonomous pathway in the regulation of oscillations. Through modulation of potent levels using an estradiol-inducible line, we have determined high Clock sensitivity at low potent concentrations, observing a positive correlation between potent levels and number of pre-branching sites. , their spacing and number of founder cells, and also in the number of lateral roots when joint treatment with auxins was performed. Likewise, the induction of potent results in the activation of genes in phase such as LOB-DOMAIN (LBD) 16, which could imply the inhibition of both the repressive activity of ARF7 in the ZO and its activating function in the Zone of differentiation (ZD). Our results through confocal laser microscopy show that CFRL in potent divide only symmetrically, being unable to generate asymmetric formative divisions, which are essential for the development of RL. To understand the signaling cascade mediated by IAA18/POTENT in the regulation of CFRL polarization and subsequent asymmetric cleavage, we transcriptionally analyzed CFRL after potent induction and co-treatment with auxin using RNAseq. In this process, we have optimized the protocols for extracting protoplasts from scarce and internal tissues such as CFRL in the pericycle. RNAseq analysis of CFRLs after potent induction and after potent induction and auxin treatment shows a total of 3811 differentially expressed genes with the potential to control some aspect of CFRL polarization and asymmetric first division. Given the role of IAA18/POTENT in auxin signaling, we analyzed its regulation on the LBDs transcription factors that play a role in the asymmetric divisions of the CFRL. Most of the differentially expressed LBDs are repressed, indicating the activation of previously described auxin response pathways along with others still unknown. Signaling by the peptides GLV6/GLV10 and TOLS2/PIP2 plays an important inhibitory role in the regulation of the anticlinal divisions of the pericycle and can be extrapolated to CFRL through the phosphorylation of MPK6 by receptor kinases, which in turn would imply downstream activity. of the factors WRKY23, PLT5 and PUCHI among others. Through the comparison between our RNAseq data and those previously described for GLV6 and TOLS2, we have determined that there is hardly any overlap between both signaling cascades. Likewise, by means of potent induction assays in the mpk6 mutant, we have determined that the regulation of the polarization and asymmetric divisions of CFRL mediated by IAA18/POTENT does not require the activity of MPK6, which confirms that said regulation is independent of GLV6. /GLV10 and TOLS2/PIP2. In order to screen for differentially expressed genes that were candidates to regulate the polarization and asymmetric divisions of CFRLs, we used the LRMap previously generated in our laboratory, which contains the expression levels of all genes throughout a chronology of the development of lateral roots in early stages. In this analysis we focus on stage I and especially on the behavior of genes between CFRL before and after nuclear migration (first morphological indication of CFRL polarization). Through the phenotypic analysis of mutants we have identified the protein KINASE FAMILY PROTEIN (KFP) 1 as necessary for the polarization and asymmetric divisions of CFRLs downstream of the IAA18/POTENT signaling cascade. Since the CFRLs in kfp1 tend to divide symmetrically, we proceeded to check the localization of the KFP1 protein and determine if there was expression in the CFRLs. KFP1 is found in most cell membranes, including CFRLs, and is not located in the membrane of cells formed by anticlinal divisions subsequent to the first asymmetric division, nor in the periclinal divisions that give rise to stage II, indicating a specificity for its regulation in the polarization of the first asymmetric division. As it is a gene downstream of auxins, we wondered if the KFP1 protein would respond to the addition of auxins. The levels of the KFP1 protein, as expected, respond positively to the exogenous addition of CFRL auxins. Finally, we have determined that the lack of asymmetry in the CFRL of the kfp1 mutant is associated with the absence of asymmetric localization of the membrane recycling process by endocytosis as well as with the delocalization of polarized markers during nuclear migration such as MAKR4 and PIN1

The Root Clock as a Signal Integrator System: Ensuring Balance for Survival

9 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP)The root system is essential for the survival of terrestrial plants, plant development, and adaptation to changing environments. The development of the root system relies on post-embryonic organogenesis and more specifically on the formation and growth of lateral roots (LR). The spacing of LR along the main root is underpinned by a precise prepatterning mechanism called the Root Clock. In Arabidopsis, the primary output of this mechanism involves the generation of periodic gene expression oscillations in a zone close to the root tip called the Oscillation Zone (OZ). Because of these oscillations, pre-branch sites (PBS) are established in the positions from which LR will emerge, although the oscillations can also possibly regulate the root wavy pattern and growth. Furthermore, we show that the Root Clock is present in LR. In this review, we describe the recent advances unraveling the inner machinery of Root Clock as well as the new tools to track the Root Clock activity. Moreover, we discuss the basis of how Arabidopsis can balance the creation of a repetitive pattern while integrating both endogenous and exogenous signals to adapt to changing environmental conditions. These signals can work as entrainment signals, but in occasions they also affect the periodicity and amplitude of the oscillatory dynamics in gene expression. Finally, we identify similarities with the Segmentation Clock of vertebrates and postulate the existence of a determination front delimiting the end of the oscillations in gene expression and initiating LR organogenesis through the activation of PBS in an ARF7 dependent-manner.This work was funded by Ministerio de Economía y Competitividad (MINECO) of Spain (grant PID2019-111523GB-I00 to MM-R) and by the “Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigacion of Spain [SEV-s-0672 (2017-2021) and CEX2020-000999-S-21-1] to MM-R through CBGP. LS-R was supported by FPI contract BES-2014-068852 from MINECO and EB-A by contract associated to Programa Atraccion de Talento from CM (2017-T2/BIO-3453, PI: P. Perez-Garcia).Peer reviewe

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

Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root Cell Populations

12 Pág.Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.This research was funded by grants from the Ministerio de Economía y Competitividad (MINECO) of Spain BIO2017-82209-R to J.C.d.P., MINECO and ERDF BFU2016-80315-P to M.A.M.-R., and by the ‘Severo Ochoa Program for Centres of Excellence in R&D’ from the Agencia Estatal de Investigacion of Spain (grantSEV-2016-0672 (2017-2021) to the CBGP. M.P.G.-G. was supported by a postdoctoral contract associated with the Severo Ochoa Program at CBGP and E.B.-A. by an Ayudante de Investigacion contract PEJ-2017-AI/BIO-7360 from Comunidad Madrid.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation grantSEV-2016-0672 (2017-2021)Peer reviewe

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis

Body regeneration through formation of new organs is a major question in developmental biology. We investigated de novo root formation using whole leaves of Arabidopsis (Arabidopsis thaliana). Our results show that local cytokinin biosynthesis and auxin biosynthesis in the leaf blade followed by auxin long-distance transport to the petiole leads to proliferation of J0121-marked xylem-associated tissues and others through signaling of INDOLE-3-ACETIC ACID INDUCIBLE28 (IAA28), CRANE (IAA18), WOODEN LEG, and ARABIDOPSIS RESPONSE REGULATORS1 (ARR1), ARR10, and ARR12. Vasculature proliferation also involves the cell cycle regulator KIP-RELATED PROTEIN2 and ABERRANT LATERAL ROOT FORMATION4, resulting in a mass of cells with rooting competence that resembles callus formation. Endogenous callus formation precedes specification of postembryonic root founder cells, from which roots are initiated through the activity of SHORT-ROOT, PLETHORA1 (PLT1), and PLT2. Primordia initiation is blocked in shr plt1 plt2 mutant. Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved