RESUMO
Auxin dictates root architecture via the Auxin Response Factor (ARF) family of transcription factors, which control lateral root (LR) formation. In Arabidopsis, ARF7 regulates the specification of prebranch sites (PBS) generating LRs through gene expression oscillations and plays a pivotal role during LR initiation. Despite the importance of ARF7 in this process, there is a surprising lack of knowledge about how ARF7 turnover is regulated and how this impacts root architecture. Here, we show that ARF7 accumulates in autophagy mutants and is degraded through NBR1-dependent selective autophagy. We demonstrate that the previously reported rhythmic changes to ARF7 abundance in roots are modulated via autophagy and might occur in other tissues. In addition, we show that the level of co-localization between ARF7 and autophagy markers oscillates and can be modulated by auxin to trigger ARF7 turnover. Furthermore, we observe that autophagy impairment prevents ARF7 oscillation and reduces both PBS establishment and LR formation. In conclusion, we report a novel role for autophagy during development, namely by enacting auxin-induced selective degradation of ARF7 to optimize periodic root branching.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Autofagia , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Raízes de Plantas , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de TransporteRESUMO
The root system plays an essential role in plant growth and adaptation to the surrounding environment. The root clock periodically specifies lateral root prebranch sites (PBS), where a group of pericycle founder cells (FC) is primed to become lateral root founder cells and eventually give rise to lateral root primordia or lateral roots (LRs). This clock-driven organ formation process is tightly controlled by modulation of auxin content and signaling. Auxin perception entails the physical interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOX (AFBs) proteins with AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to form a co-receptor system. Despite the apparent simplicity, the understanding of how specific auxin co-receptors are assembled remains unclear. We identified the compound bis-methyl auxin conjugated with N-glucoside, or BiAux, in Arabidopsis (Arabidopsis thaliana) that specifically induces the formation of PBS and the emergence of LR, with a slight effect on root elongation. Docking analyses indicated that BiAux binds to F-box proteins, and we showed that BiAux function depends on TIR1 and AFB2 F-box proteins and AUXIN RESPONSE FACTOR 7 activity, which is involved in FC specification and LR formation. Finally, using a yeast (Saccharomyces cerevisiae) heterologous expression system, we showed that BiAux favors the assemblage of specific co-receptors subunits involved in LR formation and enhances AUXIN/INDOLE-3-ACETIC ACID 28 protein degradation. These results indicate that BiAux acts as an allosteric modulator of specific auxin co-receptors. Therefore, BiAux exerts a fine-tune regulation of auxin signaling aimed to the specific formation of LR among the many development processes regulated by auxin.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Raízes de Plantas , Ácidos Indolacéticos/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Reguladores de Crescimento de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomarcadores , Diferenciação Celular , Modelos Biológicos , Mutação , Fatores de Transcrição/genéticaRESUMO
Multicellular organisms show the ability to replace damage cells, tissues and even whole organs through regeneration mechanisms. Plants show a remarkable regenerative potential. While the basic principles of plant regeneration have been known for a number of decades, the molecular and cellular mechanisms underlying such principles are currently starting to emerge. Some of these mechanisms point to the existence of highly reprogrammable cells. Developmental plasticity is a hallmark for stem cells, and stem cells are responsible for the generation of distinctive cell types forming plants. In the last years, a number of players and molecular mechanism regulating stem cell maintenance have been described, and some of them have also been involved in regenerative processes. These discoveries in plant stem cell regulation and regeneration invite us to rethink several of the classical concepts in plant biology such as cell fate specification and even the actual meaning of what we consider stem cells in plants. In this review we will cover some of these discoveries, focusing on the role of the plant stem cell function and regulation during cell and organ regeneration.
Assuntos
Desenvolvimento Vegetal/fisiologia , Regeneração/fisiologia , Reprogramação Celular , Plantas , Células-Tronco/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Flavonóis/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Diferenciação Celular/fisiologia , Diferenciação Celular/efeitos da radiação , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Luz , Fototropismo/genética , Fototropismo/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/fisiologia , Raízes de Plantas/efeitos da radiação , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiaçãoRESUMO
Stem cells are defined by their ability to self-renew and produce daughter cells that proliferate and mature. These maturing cells transition from a proliferative state to a terminal state through the process of differentiation. In the Arabidopsis thaliana root the transcription factors SCARECROW and SHORTROOT regulate specification of the bipotent stem cell that gives rise to cortical and endodermal progenitors. Subsequent progenitor proliferation and differentiation generate mature endodermis, marked by the Casparian strip, a cell-wall modification that prevents ion diffusion into and out of the vasculature. We identified a transcription factor, MYB DOMAIN PROTEIN 36 (MYB36), that regulates the transition from proliferation to differentiation in the endodermis. We show that SCARECROW directly activates MYB36 expression, and that MYB36 likely acts in a feed-forward loop to regulate essential Casparian strip formation genes. We show that myb36 mutants have delayed and defective barrier formation as well as extra divisions in the meristem. Our results demonstrate that MYB36 is a critical positive regulator of differentiation and negative regulator of cell proliferation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Raízes de Plantas/fisiologia , Fatores de Transcrição/metabolismo , Primers do DNA/genética , Mutagênese , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Meristema/citologia , Meristema/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Basidiomycota/fisiologia , Manosiltransferases/metabolismo , Peroxidases/metabolismo , beta-Glucosidase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Divisão Celular , Glicosilação , Concentração de Íons de Hidrogênio , Íntrons/genética , Manosiltransferases/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos da radiação , Mutação , Peroxidases/genética , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Proteômica , Splicing de RNA , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , beta-Glucosidase/genéticaRESUMO
In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.
Assuntos
Escuridão , Luz , Raízes de Plantas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Raízes de Plantas/fisiologia , Raízes de Plantas/efeitos da radiaçãoRESUMO
A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.
Assuntos
Arabidopsis/metabolismo , Linhagem da Célula , Dosagem de Genes , MicroRNAs/metabolismo , Raízes de Plantas/citologia , RNA de Plantas/metabolismo , Transdução de Sinais , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Padronização Corporal , Movimento Celular , Endoderma/citologia , Endoderma/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Organogênese , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transporte de RNA , RNA de Plantas/genética , Fatores de Transcrição/metabolismo , Xilema/citologia , Xilema/crescimento & desenvolvimento , Xilema/metabolismoRESUMO
Plant growth and development involve the specification and regeneration of stem cell niches (SCNs). Although plants are exposed to disparate environmental conditions, how environmental cues affect developmental programs and stem cells is not well understood. Root stem cells are accommodated in meristems in SCNs around the quiescent center (QC), which maintains their activity. Using a combination of genetics and confocal microscopy to trace morphological defects and correlate them with changes in gene expression and protein levels, we show that the cold-induced transcription factor (TF) C-REPEAT BINDING FACTOR 3 (CBF3), which has previously been associated with cold acclimation, regulates root development, stem cell activity, and regeneration. CBF3 is integrated into the SHORT-ROOT (SHR) regulatory network, forming a feedback loop that maintains SHR expression. CBF3 is primarily expressed in the root endodermis, whereas the CBF3 protein is localized to other meristematic tissues, including root SCNs. Complementation of cbf3-1 using a wild-type CBF3 gene and a CBF3 fusion with reduced mobility show that CBF3 movement capacity is required for SCN patterning and regulates root growth. Notably, cold induces CBF3, affecting QC activity. Furthermore, exposure to moderate cold around 10°C-12°C promotes root regeneration and QC respecification in a CBF3-dependent manner during the recuperation period. By contrast, CBF3 does not appear to regulate stem cell survival, which has been associated with recuperation from more acute cold (â¼4°C). We propose a role for CBF3 in mediating the molecular interrelationships among the cold response, stem cell activity, and development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plantas/metabolismo , Células-TroncoRESUMO
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.
RESUMO
Oscillatory mechanisms are present in most life forms and regulate biological processes periodically. In multicellular organisms where more than one oscillatory mechanism is present, they are organized forming a hierarchical coordinated system even at the cellular level. Here, we focus on the Root Clock, an oscillatory mechanism located at the tip of roots that patterns the spacing of lateral organs through oscillating gene expression. We present a series of recent findings and hypotheses about the cellular mechanisms driving the oscillations, how oscillatory information is transmitted within this clock and similarities with other oscillatory systems. Next, we review principles of communication in other pulsatile mechanisms such as circadian rhythms in plants and mammals, and address the possible communication between plant circadian rhythms and the Root Clock. Finally, we advocate for the use of single-cell approaches to address cell communication, synchronization and integration of external outputs into the Root Clock system.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Comunicação Celular , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Mamíferos/fisiologiaRESUMO
Over the last decades, research on postembryonic root development has been facilitated by "omics" technologies. Among these technologies, microarrays first, and RNA sequencing (RNA-seq) later, have provided transcriptional information on the underlying molecular processes establishing the basis of System Biology studies in roots. Cell fate specification and development have been widely studied in the primary root, which involved the identification of many cell type transcriptomes and the reconstruction of gene regulatory networks (GRN). The study of lateral root (LR) development has not been an exception. However, the molecular mechanisms regulating cell fate specification during LR formation remain largely unexplored. Recently, single-cell RNA-seq (scRNA-seq) studies have addressed the specification of tissues from stem cells in the primary root. scRNA-seq studies are anticipated to be a useful approach to decipher cell fate specification and patterning during LR formation. In this review, we address the different scRNA-seq strategies used both in plants and animals and how we could take advantage of scRNA-seq to unravel new regulatory mechanisms and reconstruct GRN. In addition, we discuss how to integrate scRNA-seq results with previous RNA-seq datasets and GRN. We also address relevant findings obtained through single-cell based studies and how LR developmental studies could be facilitated by scRNA-seq approaches and subsequent GRN inference. The use of single-cell approaches to investigate LR formation could help to decipher fundamental biological mechanisms such as cell memory, synchronization, polarization, or pluripotency.
RESUMO
Postembryonic organogenesis is critical for plant development. Underground, lateral roots (LRs) form the bulk of mature root systems, yet the ontogeny of the LR primordium (LRP) is not clear. In this study, we performed the single-cell RNA sequencing through the first four stages of LR formation in Arabidopsis. Our analysis led to a model in which a single group of precursor cells, with a cell identity different from their pericycle origins, rapidly reprograms and splits into a mixed ground tissue/stem cell niche fate and a vascular precursor fate. The ground tissue and stem cell niche fates soon separate and a subset of more specialized vascular cells form sucrose transporting phloem cells that appear to connect to the primary root. We did not detect cells resembling epidermis or root cap, suggesting that outer tissues may form later, preceding LR emergence. At this stage, some remaining initial precursor cells form the primordium flanks, while the rest create a reservoir of pluripotent cells that are able to replace the LR if damaged. Laser ablation of the central and lateral LRP regions showed that remaining cells restart the sequence of tissue initiation to form a LR. Collectively, our study reveals an ontological hierarchy for LR formation with an early and sequential split of main root tissues and stem cells.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Organogênese Vegetal/genética , Desenvolvimento Vegetal/genética , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/citologia , Raízes de Plantas/citologia , Análise de Sequência de RNA , Células-Tronco/citologiaRESUMO
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.
RESUMO
In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Relógios Biológicos/genética , Parede Celular/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Pectinas/metabolismo , Raízes de Plantas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Esterificação/genética , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , NADPH Oxidases/metabolismo , Raízes de Plantas/genética , Vesículas Transportadoras/fisiologiaRESUMO
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.
RESUMO
Root-knot nematodes (RKNs; Meloidogyne spp.) induce feeding cells (giant cells; GCs) inside a pseudo-organ (gall) from still unknown root cells. Understanding GCs ontogeny is essential to the basic knowledge of RKN-plant interaction and to discover novel and effective control strategies. Hence, we report for the first time in a model plant, Arabidopsis, molecular, and cellular features concerning ectopic de novo organogenesis of RKNs GCs in leaves. RKNs induce GCs in leaves with irregular shape, a reticulated cytosol, and fragmented vacuoles as GCs from roots. Leaf cells around the nematode enter G2-M shown by ProCycB1;1:CycB1;1(NT)-GUS expression, consistent to multinucleated GCs. In addition, GCs nuclei present irregular and varied sizes. All these characteristics mentioned, being equivalent to GCs in root-galls. RKNs complete their life cycle forming a gall/callus-like structure in the leaf vascular tissues resembling auxin-induced callus with an auxin-response maxima, indicated by high expression of DR5::GUS that is dependent on leaf auxin-transport. Notably, induction of leaves calli/GCs requires molecular components from roots crucial for lateral roots (LRs), auxin-induced callus and root-gall formation, i.e., LBD16. Hence, LBD16 is a xylem pole pericycle specific and local marker in LR primordia unexpectedly induced locally in the vascular tissue of leaves after RKN infection. LBD16 is also fundamental for feeding site formation as RKNs could not stablish in 35S::LBD16-SRDX leaves, and likely it is also a conserved molecular hub between biotic and developmental signals in Arabidopsis either in roots or leaves. Moreover, RKNs induce the ectopic development of roots from leaf and root-galls, also formed in mutants compromised in LR formation, arf7/arf19, slr, and alf4. Therefore, nematodes must target molecular signatures to induce post-embryogenic de novo organogenesis through the LBD16 callus formation pathway partially different from those prevalent during normal LR development.
RESUMO
Tissue patterns are dynamically maintained. Continuous formation of plant tissues during postembryonic growth requires asymmetric divisions and the specification of cell lineages. We show that the BIRDs and SCARECROW regulate lineage identity, positional signals, patterning, and formative divisions throughout Arabidopsis root growth. These transcription factors are postembryonic determinants of the ground tissue stem cells and their lineage. Upon further activation by the positional signal SHORT-ROOT (a mobile transcription factor), they direct asymmetric cell divisions and patterning of cell types. The BIRDs and SCARECROW with SHORT-ROOT organize tissue patterns at all formative steps during growth, ensuring developmental plasticity.