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[EN] Adverse environmental conditions trigger responses in plants that promote stress tolerance and survival at the expense of growth(1). However, little is known of how stress signalling pathways interact with each other and with growth regulatory components to balance growth and stress responses. Here, we show that plant growth is largely regulated by the interplay between the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1) protein kinase and the abscisic acid (ABA) phytohormone pathway. While SnRK2 kinases are main drivers of ABA-triggered stress responses, we uncover an unexpected growth-promoting function of these kinases in the absence of ABA as repressors of SnRK1. Sequestration of SnRK1 by SnRK2-containing complexes inhibits SnRK1 signalling, thereby allowing target of rapamycin (TOR) activity and growth under optimal conditions. On the other hand, these complexes are essential for releasing and activating SnRK1 in response to ABA, leading to the inhibition of TOR and growth under stress. This dual regulation of SnRK1 by SnRK2 kinases couples growth control with environmental factors typical for the terrestrial habitat and is likely to have been critical for the water-to-land transition of plants., We thank J.-K. Zhu for the snrk2 mutants, M. Bennett for the SnRK2.2-GFP line, C. Koncz for the SnRK1-GFP line, X. Li for the SnRK2.3-FLAG OE line, J. Schroeder for the GFP-His-FLAG and SnRK2.6-His-FLAG OE lines, C. Mackintosh for the TPS5 antibody and the Nottingham Arabidopsis stock centre for T-DNA mutant seeds. The IGC Plant Facility (Vera Nunes) is thanked for excellent plant care. This work was supported by Fundacao para a Ciencia e a Tecnologia through the R&D Units UIDB/04551/2020 (GREEN-IT-Bioresources for Sustainability) and UID/MAR/04292/2019, FCT project nos. PTDC/BIA-PLA/7143/2014, LISBOA-01-0145-FEDER-028128 and PTDC/BIA-BID/32347/2017, and FCT fellowships/contract nos. SFRH/BD/122736/2016 (M.A.), SFRH/BPD/109336/2015 (A.C.), PD/BD/150239/2019 (D.R.B.), and IF/00804/2013 (E.B.G.). Work in P.L.R.'s laboratory was funded by MCIU grant no. BIO2017-82503-R. C.M. thanks the LabEx Paris Saclay Plant Sciences-SPS (ANR-10-LABX-040-SPS) for support. B.B.P. was funded by Programa VALi+d GVA APOSTD/2017/039. This project has received funding from the European Union Horizon 2020 research and innovation programme (grant agreement no. 867426-ABA-GrowthBalance-H2020-WF-2018-2020/H2020-WF-01-2018, awarded to B.B.P.). This work is dedicated to the memory of our beloved friend and colleague Americo Rodrigues.

The evolutionarily conserved protein kinase complexes SnRK1 and TOR are central metabolic regulators essential for plant growth, development, and stress responses. They are activated by opposite signals, and the outcome of their activation is, in global terms, antagonistic. Similarly to their yeast and animal counterparts, SnRK1 is activated by the energy deficit often associated with stress to restore homeostasis, while TOR is activated in nutrient-rich conditions to promote growth. Recent evidence suggests that SnRK1 represses TOR in plants, revealing evolutionary conservation also in their crosstalk. Given their importance for integrating environmental information into growth and developmental programs, these signaling pathways hold great promise for reducing the growth penalties caused by stress. Here we review the literature connecting SnRK1 and TOR to plant stress responses. Although SnRK1 and TOR emerge mostly as positive regulators of defense and growth, respectively, the outcome of their activities in plant growth and performance is not always straightforward. Manipulation of both pathways under similar experimental setups, as well as further biochemical and genetic analyses of their molecular and functional interaction, is essential to fully understand the mechanisms through which these two metabolic pathways contribute to stress responses, growth, and development. info:eu-repo/semantics/publishedVersion

Arabidopsis mesophyll protoplasts can be readily isolated and transfected in order to transiently express proteins of interest. As freshly isolated mesophyll protoplasts maintain essentially the same physiological characteristics of whole leaves, this cell-based transient expression system can be used to molecularly dissect the responses to various stress conditions. The response of stress-responsive promoters to specific stimuli can be accessed via reporter gene assays. Additionally, reporter systems can be easily engineered to address other levels of regulation, such as transcript and/or protein stability. Here we present a detailed protocol for using the Arabidopsis mesophyll protoplast system to study responses to environmental stress, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.

Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants., NF is funded by an FI PhD fellowship from the Generalitat de Catalunya. PFJ acknowledges the FPU grant (FPU-AP2008-03325) funded by the Spanish Ministry of Education (2009–2011) and the Spanish Ministry of Education, Culture and Sports (2011–2013). AC is funded by a post-doctoral fellowship from Fundação para a Ciência e Tecnologia (SFRH/BPD/47280/2008). AICD is a recipient of a Marie-Curie Initial Training Network “BRAVISSIMO” (Grant PITN-GA- 2008- 215118). This work is supported by the Spanish Ministry of Science and Innovation through grants FIS2012-37655-C02-02 (PFJ and MI), FIS2009-13360-C03-01 (PFJ and MI), BIO2010-16673 (NF, AC and AICD) and BIO2013-43873 Grant Excellence to AICD, the Generalitat de Catalunya through grants 2009 SGR 0014 and 2014 SGR 878 (PFJ and MI), the Academy of Finland and the University of Helsinki (RSi and APM), Integrative Life Science Doctoral Program (RSi) and National Science Foundation Grant DBI0820755 (JMA). RSw. and MJB acknowledge the BBSRC and EPSRC for funding.

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo. Fundação para a Ciência e Tecnologia fellowships: (SFRH/BD/33563/2008, SFRH/BPD/47280/2008, SFRH/BPD/79255/2011) and grant: (PTCD/BIA-BCM/107924/2008); EMBO fellowship & EMBO Installation program; Deutsche Forschungsgemeinschaft grant: (SPP1530); Max Planck Society grant.

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways., E.B.-G. was supported by grants from Marie Curie IRG, the EMBO Installation program, Marie Curie Actions FP7-People-2010-ITN, the Fundação para a Ciência e a Tecnologia (FCT-PTDC/AGR-AAM/104939/2008), and the Portugal-Spain Bilateral Collaboration program Ações integradas (Ação E-26/10). A.C. was supported by SFRH/BPD/47280/2008, C.M. was supported by SFRH/BD/33563/2008, L.M. was supported by SFRH/BD/51627/2011, and P.C. was supported by SFRH/BPD/79255/2011. A. Rabissi was supported by a Generalitat de Catalunya PhD grant (FI-AR067443). P.L.R. was supported by the Ministerio de Ciencia e Innovación (grants BIO2011-23446 and PT2009-0155), R.A. was supported by the Junta para Ampliación de Estudios e Investigaciones Científicas-Consejo Superior de Investigaciones Cientificas fellowship, and M.G.-G. was supported by a Juan de la Cierva contract.

During stomatal closure, nitric oxide (NO) operates as one of the key intermediates in the complex, abscisic acid (ABA)-mediated, guard cell signaling network that regulates this process. However, data concerning the role of NO in stomatal closure that occurs in turgid vs. dehydrated plants is limited. The data presented demonstrate that, while there is a requirement for NO during the ABA-induced stomatal closure of turgid leaves, such a requirement does not exist for ABA-enhanced stomatal closure observed to occur during conditions of rapid dehydration. The data also indicate that the ABA signaling pathway must be both functional and to some degree activated for guard cell NO signaling to occur. These observations are in line with the idea that the effects of NO in guard cells are mediated via a Ca2+-dependent rather than a Ca2+-independent ABA signaling pathway. It appears that there is a role for NO in the fine tuning of the stomatal apertures of turgid leaves that occurs in response to fluctuations in the prevailing environment.

Plants contain multiple forms of catalase (CAT) and their specific functions remain uncertain. We cloned two potato cDNAs corresponding to CAT1 and CAT2 genes, analysed their phylogenetic relationship, and studied their expression and activity in different organs to gain clues to their functions. Phylogenetic trees and the alignment of CAT cDNA sequences provided evidence that CAT1 and CAT2 genes have high identity to catalases of other solanaceous species, but are not phylogenetically closely related to one another, which contradicts the phylogenetic closeness ascribed to these genes. Northern blot analyses revealed that expression of CAT genes is controlled by leaf developmental phase. CAT2 expression was higher in both very young and senescent leaves, whereas CAT1 mRNA accumulated mainly in mature leaf, where the lowest CAT2 expression occurred. CAT1 and CAT2 are also differentially expressed in root, sprout and petal. Expression and activity patterns are consistent with different physiological roles for CAT1 and CAT2 isoforms. CAT1 is considered to be associated with photorespiration whereas CAT2 would fulfill physiological roles unrelated to this process. CAT2 appears to be a multifunctional isoform, associated with glyoxysomal activity in leaf senescence, other processes in non-photosynthetic organs and defence, functions that in other solanaceous species are fulfilled by two different isoforms.