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Climate change is increasing the frequency of extreme heat events that aggravate its negative impact on plant development and agricultural yield. Most of the 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 and in a temperature descendent-gradient environment. Excessively high temperatures severely reduce cell division in the root meristem, compromising root growth, while increase cell division of quiescent center cells, likely in an attempt to maintain the stem cell niche in such harsh conditions. Here, we engineered the TGRooZ, a device that generates a temperature gradient for in vitro or greenhouse growth assays. The root system of plants exposed to high temperatures in the shoot, but cultivated in the TGRooZ, grows efficiently and maintains its functionality to sustain a proper shoot growth and development. Furthermore, gene expression and rhizosphere or root microbiomes composition of TGRooZ-grown roots is significantly less affected than high temperature-grown roots, correlating with a higher root functionality. Our data indicate that by using TGRooZ in heat stress studies, we can improve our knowledge on the plant response to high temperatures and the applicability from laboratory studies to the field.

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, This work was supported by the MINECO BIO2012-32796 Spanish grant, the Juan de la Cierva Fellowship Program (JCI-2012-14172) (MINECO, Spain) (to M.R), the Postgraduate Studies Scholarship Programme (UAM, Spain) (to L.P.V.) and the FPU Fellowship Program (AP2010-4786) (MECD, Spain) (to I.A)

© 2019 Contreras, Kallemi, González-García, Lazarova, Sánchez-Serrano, Sanmartín and Rojo., 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., This work was supported by the Spanish Ministry of Economy & Competitivity and FEDER funds (BIO2018-094257-B MICINN/FEDER to ER, MS and JJSS). RC and AL were recipient of a FPI scholarships from the Spanish Ministry of Economy and Competitivity. PK was recipient of an ERASMUS fellowship. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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.

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., M.I. acknowledges support from the Ministerio de Economía y Competitividad (Spain) and Fondo Europeo de Desarrollo Regional FEDER (EU) through grant FIS2015-66503-C3-3-P (MINECO/FEDER), from MINECO through FIS2012-37655-C02-02 and FIS2009-13360-C03-01, and from the Generalitat de Catalunya through Grup de Recerca Consolidat 2014 SGR 878. A.I.C.-D. is a recipient of BIO2016-78955 and BIO2013-43873 grants from the Spanish Ministry of Economy and Competitiveness (MINECO), of an ERC Consolidator Grant (ERC-2015-CoG—683163) from the European Research Council, and has funding from Developing an European American NGS Network (DEANN) from FP7-PEOPLE-2013-IRSES (2015–2017). M.G.-G. was the recipient of a postdoctoral contract from BIO2010-16673 grant from the Spanish Ministry of Economy and Competitiveness, J.V.B was funded by FI PhD fellowship from Generalitat de Catalunya, and I.P was funded by a JAE-CSIC PhD fellowship in A. I.C.-D. laboratory. A.P.-R. is a recipient of a PhD fellowship from the “Severo Ochoa Programme for Centers of Excellence in R&D” 2016–2019 (SEV-2015-0533)” in A.I.C-D. laboratory.

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. Fil: Muñoz, Alfonso. Consejo Superior de Investigaciones Cientificas; España Fil: Mangano, Silvina. Consejo Superior de Investigaciones Cientificas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: González-García, Mary Paz. Consejo Superior de Investigaciones Cientificas; España Fil: Contreras, Ramón. Consejo Superior de Investigaciones Cientificas; España Fil: Sauer, Michael. Universitat Potsdam; Alemania. Consejo Superior de Investigaciones Cientificas; España Fil: De Rybel, Bert. University of Ghent; Bélgica. Wageningen University; Países Bajos Fil: Weijers, Dolf. Wageningen University; Países Bajos Fil: Sánchez Serrano, José Juan. Consejo Superior de Investigaciones Cientificas; España Fil: Sanmartín, Maite. Consejo Superior de Investigaciones Cientificas; España Fil: Rojo, Enrique. Consejo Superior de Investigaciones Cientificas; España

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., This work was supported by the FP7 Marie-Curie Initial Training Network ‘BRAVISSIMO’ (PITN-GA-2008-215118; E.R. and A.C-D), the Belgian Science Policy (BELSPO) (M.K.Z.) and the Agency for Innovation by Science and Technology (IWT) (who awarded a postdoctoral fellowship to M.K.Z., a pre-doctoral fellowship to S.D. and a ‘Strategisch Basisonderzoek’ grant (no. 60839) to C.B.). A.C-D. receives funding through grants from the Spanish Ministry of Science and Innovation (BIO2008/00505).

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.

Mapping centromere distribution in complete organs is a key step towards establishing how this couples to organ development and cell functions. In this context, quantitative microscopy tools play a key role, as they allow a precise measurement of centromere presence at both individual cell and whole organ levels. This work introduces PloidyQuantX, an imaging tool that operates on confocal microscopy image stacks. Tested on imagery obtained from whole-mount centromere Q-FISH of the Arabidopsis thaliana primary root, PloidyQuantX incorporates interactive segmentation and image analysis modules that allow quantifying the number of centromeres present in each cell nucleus, thus creating maps of centromere distribution in cells across the whole organ. Moreover, the presented tool also allows rendering three-dimensional models of each individual root cell, which makes it possible to relate their internal topology to specific cell functions.

Several gibberellic acid (GA3)-induced cDNA fragments encoding putative serine/threonine protein phosphatase (PP) 2A catalytic subunits were obtained by means of differential reverse transcriptase-PCR approach. The full-length clone, named FsPP2A1, isolated from a beechnut cDNA library, exhibited all the features of and homology to members of the PP2A family. By transient expression of FsPP2A1 in tobacco and Arabidopsis cells as a green fluorescent fusion protein, we have obtained evidence supporting the subcellular localization of this protein in both the cytosol and the nucleus. Analysis of FsPP2A1 expression during seed stratification shows that these transcripts increase in the presence of GA3, a treatment proved to be efficient in breaking the dormancy of Fagus sylvatica seeds, but they are almost undetectable in dormant seeds or when dormancy is maintained after treatment with either abscisic acid or the gibberellin biosynthesis inhibitor paclobutrazol. The PP inhibitor okadaic acid (OKA) has a clear effect in decreasing both seed germination and FsPP2A1 expression. Furthermore, FsPP2A1 is specifically expressed in seed tissues, not being detected in other vegetative tissues examined. These results show the regulation of this PP by GA3 and OKA in these seeds. Its relationship with the processes taking place during the transition from dormancy to germination is also discussed.

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.

Referred to by: Josep Vilarrasa-Blasi, Mary-Paz González-García, David Frigola, Norma Fàbregas-Vallvé, Konstantinos G. Alexiou, Nuria López-Bigas, Susana Rivas, Alain Jauneau, Jan U. Lohmann, Philip N. Benfey, Marta Ibañes, Ana I. Caño-Delgado Regulation of Plant Stem Cell Quiescence by a Brassinosteroid Signaling Module Developmental Cell, Volume 33, Issue 2, 20 April 2015, Pages 238., 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., J.V.-B. and N.F.-V. are funded by FI PhD fellowship from the Generalitat de Catalunya (GC) in the A.I.C.-D. laboratory. J.V.-B. received a short-term fellowship (BE1-00924) in the Lohmann (J.U.L.) laboratory supported by the SFB873 of the DFG. Research by D.F and M.I. is funded by FIS2012-37655-C02-02 by the Spanish Ministry de Economy and Competitiveness and 2009SGR14 from GC, and D.F. has a PhD fellowship (FPU-AP2009-3736). S.R. is funded by the Laboratoire d’Excellence (LABEX) TULIP (ANR-10-LABX-41). M.-P.G.-G. received a “Juan de la Cierva” postdoctoral contract from the Spanish Ministry of Science in the Ana Caño (A.I.C.-D.) laboratory, and an HFSP short-term fellowship in the Benfey (P.N.B.) laboratory. P.N.B. is funded by NSF Arabidopsis 2010 grant. Work in the Ana Caño (A.I.C.-D.) laboratory is funded by a BIO2010/007 grant from the Spanish Ministry of Innovation and Science and a Marie-Curie Initial Training Network “BRAVISSIMO” (grant no. PITN-GA-2008-215118).

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.