Synthetically derived BiAux modulates auxin co-receptor activity to stimulate lateral root formation.

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 LR founder cells (LRFCs) and eventually give rise to lateral root primordia (LRP) 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 (ARF7) 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 (IAA28) 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.

Temperature changes in the root ecosystem affect plant functionality.

Climate change is increasing the frequency of extreme heat events that aggravate its negative impact on plant development and agricultural yield. Most experiments designed to study plant adaption to heat stress apply homogeneous high temperatures to both shoot and root. However, this treatment does not mimic the conditions in natural fields, where roots grow in a dark environment with a descending temperature gradient. Excessively high temperatures severely decrease cell division in the root meristem, compromising root growth, while increasing the division of quiescent center cells, likely in an attempt to maintain the stem cell niche under such harsh conditions. Here, we engineered the TGRooZ, a device that generates a temperature gradient for in vitro or greenhouse growth assays. The root systems of plants exposed to high shoot temperatures but cultivated in the TGRooZ grow efficiently and maintain their functionality to sustain proper shoot growth and development. Furthermore, gene expression and rhizosphere or root microbiome composition are significantly less affected in TGRooZ-grown roots than in high-temperature-grown roots, correlating with higher root functionality. Our data indicate that use of the TGRooZ in heat-stress studies can improve our knowledge of plant response to high temperatures, demonstrating its applicability from laboratory studies to the field.

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

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.

Identification of Domains and Factors Involved in MINIYO Nuclear Import.

The transition of stem cells from self-renewal into differentiation is tightly regulated to assure proper development of the organism. Arabidopsis MINIYO (IYO) and its mammalian orthologue RNA polymerase II associated protein 1 (RPAP1) are essential factors for initiating stem cell differentiation in plants and animals. Moreover, there is evidence suggesting that the translocation of IYO and RPAP1 from the cytosol into the nucleus functions as a molecular switch to initiate this cell fate transition. Identifying the determinants of IYO subcellular localization would allow testing if, indeed, nuclear IYO migration triggers cell differentiation and could provide tools to control this crucial developmental transition. Through transient and stable expression assays in Nicotiana benthamiana and Arabidopsis thaliana, we demonstrate that IYO contains two nuclear localization signals (NLSs), located at the N- and C-terminus of the protein, which mediate the interaction with the NLS-receptor IMPA4 and the import of the protein into the nucleus. Interestingly, IYO also interacts with GPN GTPases, which are involved in selective nuclear import of RNA polymerase II. This interaction is prevented when the G1 motif in GPN1 is mutated, suggesting that IYO binds specifically to the nucleotide-bound form of GPN1. In contrast, deleting the NLSs in IYO does not prevent the interaction with GPN1, but it interferes with import of GPN1 into the nucleus, indicating that IYO and GPN1 are co-transported as a complex that requires the IYO NLSs for import. This work unveils key domains and factors involved in IYO nuclear import, which may prove instrumental to determine how IYO and RPAP1 control stem cell differentiation.

The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms.

The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL85KR mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.

Boron deficiency inhibits root growth by controlling meristem activity under cytokinin regulation.

Significant advances have been made in the last years trying to identify regulatory pathways that control plant responses to boron (B) deficiency. Still, there is a lack of a deep understanding of how they act regulating growth and development under B limiting conditions. Here, we analyzed the impact of B deficit on cell division leading to root apical meristem (RAM) disorganization. Our results reveal that inhibition of cell proliferation under the regulatory control of cytokinins (CKs) is an early event contributing to root growth arrest under B deficiency. An early recovery of QC46:GUS expression after transferring B-deficient seedlings to control conditions revealed a role of B in the maintenance of QC identity whose loss under deficiency occurred at later stages of the stress. Additionally, the D-type cyclin CYCD3 overexpressor and triple mutant cycd3;1-3 were used to evaluate the effect on mitosis inhibition at the G1-S boundary. Overall, this study supports the hypothesis that meristem activity is inhibited by B deficiency at early stages of the stress as it does cell elongation. Likewise, distinct regulatory mechanisms seem to take place depending on the severity of the stress. The results presented here are key to better understand early signaling responses under B deficiency.

A Sizer model for cell differentiation in Arabidopsis thaliana root growth.

Plant roots grow due to cell division in the meristem and subsequent cell elongation and differentiation, a tightly coordinated process that ensures growth and adaptation to the changing environment. How the newly formed cells decide to stop elongating becoming fully differentiated is not yet understood. To address this question, we established a novel approach that combines the quantitative phenotypic variability of wild-type Arabidopsis roots with computational data from mathematical models. Our analyses reveal that primary root growth is consistent with a Sizer mechanism, in which cells sense their length and stop elongating when reaching a threshold value. The local expression of brassinosteroid receptors only in the meristem is sufficient to set this value. Analysis of roots insensitive to BR signaling and of roots with gibberellin biosynthesis inhibited suggests distinct roles of these hormones on cell expansion termination. Overall, our study underscores the value of using computational modeling together with quantitative data to understand root growth.

RIMA-Dependent Nuclear Accumulation of IYO Triggers Auxin-Irreversible Cell Differentiation in Arabidopsis.

The transcriptional regulator MINIYO (IYO) is essential and rate-limiting for initiating cell differentiation in Arabidopsis thaliana Moreover, IYO moves from the cytosol into the nucleus in cells at the meristem periphery, possibly triggering their differentiation. However, the genetic mechanisms controlling IYO nuclear accumulation were unknown, and the evidence that increased nuclear IYO levels trigger differentiation remained correlative. Searching for IYO interactors, we identified RPAP2 IYO Mate (RIMA), a homolog of yeast and human proteins linked to nuclear import of selective cargo. Knockdown of RIMA causes delayed onset of cell differentiation, phenocopying the effects of IYO knockdown at the transcriptomic and developmental levels. Moreover, differentiation is completely blocked when IYO and RIMA activities are simultaneously reduced and is synergistically accelerated when IYO and RIMA are concurrently overexpressed, confirming their functional interaction. Indeed, RIMA knockdown reduces the nuclear levels of IYO and prevents its prodifferentiation activity, supporting the conclusion that RIMA-dependent nuclear IYO accumulation triggers cell differentiation in Arabidopsis. Importantly, by analyzing the effect of the IYO/RIMA pathway on xylem pole pericycle cells, we provide compelling evidence reinforcing the view that the capacity for de novo organogenesis and regeneration from mature plant tissues can reside in stem cell reservoirs.

Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants.

Regulation of plant stem cell quiescence by a brassinosteroid signaling module.

The quiescent center (QC) maintains the activity of the surrounding stem cells within the root stem cell niche, yet specific molecular players sustaining the low rate of QC cell division remain poorly understood. Here, we identified a R2R3-MYB transcription factor, BRAVO (BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER), acting as a cell-specific repressor of QC divisions in the primary root of Arabidopsis. Ectopic BRAVO expression restricts overall root growth and ceases root regeneration upon damage of the stem cells, demonstrating the role of BRAVO in counteracting Brassinosteroid (BR)-mediated cell division in the QC cells. Interestingly, BR-regulated transcription factor BES1 (BRI1-EMS SUPRESSOR 1) directly represses and physically interacts with BRAVO in vivo, creating a switch that modulates QC divisions at the root stem cell niche. Together, our results define a mechanism for BR-mediated regulation of stem cell quiescence in plants.

Brassinosteroid production and signaling differentially control cell division and expansion in the leaf.

Brassinosteroid (BR) hormones control plant growth through acting on both cell expansion and division. Here, we examined the role of BRs in leaf growth using the Arabidopsis BR-deficient mutant constitutive photomorphogenesis and dwarfism (cpd). We show that the reduced size of cpd leaf blades is a result of a decrease in cell size and number, as well as in venation length and complexity. Kinematic growth analysis and tissue-specific marker gene expression revealed that the leaf phenotype of cpd is associated with a prolonged cell division phase and delayed differentiation. cpd-leaf-rescue experiments and leaf growth analysis of BR biosynthesis and signaling gain-of-function mutants showed that BR production and BR receptor-dependent signaling differentially control the balance between cell division and expansion in the leaf. Investigation of cell cycle markers in leaves of cpd revealed the accumulation of mitotic proteins independent of transcription. This correlated with an increase in cyclin-dependent kinase activity, suggesting a role for BRs in control of mitosis.

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots.

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.

The nuclear interactor PYL8/RCAR3 of Fagus sylvatica FsPP2C1 is a positive regulator of abscisic acid signaling in seeds and stress.

The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses.

Overexpression of a protein phosphatase 2C from beech seeds in Arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis.

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis.

Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling.

HAB1 was originally cloned on the basis of sequence homology to ABI1 and ABI2, and indeed, a multiple sequence alignment of 32 Arabidopsis protein phosphatases type-2C (PP2Cs) reveals a cluster composed by the four closely related proteins, ABI1, ABI2, HAB1 and At1g17550 (here named HAB2). Characterisation of transgenic plants harbouring a transcriptional fusion ProHAB1: green fluorescent protein (GFP) indicates that HAB1 is broadly expressed within the plant, including key target sites of abscisic acid (ABA) action as guard cells or seeds. The expression of the HAB1 mRNA in vegetative tissues is strongly upregulated in response to exogenous ABA. In this work, we show that constitutive expression of HAB1 in Arabidopsis under a cauliflower mosaic virus (CaMV) 35S promoter led to reduced ABA sensitivity both in seeds and vegetative tissues, compared to wild-type plants. Thus, in the field of ABA signalling, this work represents an example of a stable phenotype in planta after sustained overexpression of a PP2C genes. Additionally, a recessive T-DNA insertion mutant of HAB1 was analysed in this work, whereas previous studies of recessive alleles of PP2C genes were carried out with intragenic revertants of the abi1-1 and abi2-1 mutants that carry missense mutations in conserved regions of the PP2C domain. In the presence of exogenous ABA, hab1-1 mutant shows ABA-hypersensitive inhibition of seed germination; however, its transpiration rate was similar to that of wild-type plants. The ABA-hypersensitive phenotype of hab1-1 seeds together with the reduced ABA sensitivity of 35S:HAB1 plants are consistent with a role of HAB1 as a negative regulator of ABA signalling. Finally, these results provide new genetic evidence on the function of a PP2C in ABA signalling.

Negative regulation of abscisic acid signaling by the Fagus sylvatica FsPP2C1 plays a role in seed dormancy regulation and promotion of seed germination.

FsPP2C1 was previously isolated from beech (Fagus sylvatica) seeds as a functional protein phosphatase type-2C (PP2C) with all the conserved features of these enzymes and high homology to ABI1, ABI2, and PP2CA, PP2Cs identified as negative regulators of ABA signaling. The expression of FsPP2C1 was induced upon abscisic acid (ABA) treatment and was also up-regulated during early weeks of stratification. Furthermore, this gene was specifically expressed in ABA-treated seeds and was hardly detectable in vegetative tissues. In this report, to provide genetic evidence on FsPP2C1 function in seed dormancy and germination, we used an overexpression approach in Arabidopsis because transgenic work is not feasible in beech. Constitutive expression of FsPP2C1 under the cauliflower mosaic virus 35S promoter confers ABA insensitivity in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Additionally, transgenic 35S:FsPP2C1 plants are able to germinate under unfavorable conditions, as inhibitory concentrations of mannitol, NaCl, or paclobutrazol. In vegetative tissues, Arabidopsis FsPP2C1 transgenic plants show ABA-resistant early root growth and diminished induction of the ABA-response genes RAB18 and KIN2, but no effect on stomatal closure regulation. Seed and vegetative phenotypes of Arabidopsis 35S:FsPP2C1 plants suggest that FsPP2C1 negatively regulates ABA signaling. The ABA inducibility of FsPP2C1 expression, together with the transcript accumulation mainly in seeds, suggest that it could play an important role modulating ABA signaling in beechnuts through a negative feedback loop. Finally, we suggest that negative regulation of ABA signaling by FsPP2C1 is a factor contributing to promote the transition from seed dormancy to germination during early weeks of stratification.