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The plasma membrane proton (H+)-ATPases of plants generate steep electrochemical gradients and activate osmotic solute uptake. H+-ATPase-mediated proton pumping orchestrates cellular homeostasis and is a prerequisite for plastic cell expansion and plant growth. All evidence suggests that the population of H+-ATPase proteins at the plasma membrane reflects a balance of their roles in exocytosis, endocytosis, and recycling. Auxin governs both traffic and activation of the plasma membrane H+-ATPase proteins already present at the membrane. As in other eukaryotes, in plants, SNARE-mediated membrane traffic influences the density of several proteins at the plasma membrane. Even so, H+-ATPase traffic, its relationship with SNAREs, and its regulation by auxin have remained enigmatic. Here, we identify the Arabidopsis (Arabidopsis thaliana) Qa-SNARE SYP132 (Syntaxin of Plants132) as a key factor in H+-ATPase traffic and demonstrate its association with endocytosis. SYP132 is a low-abundant, secretory SNARE that primarily localizes to the plasma membrane. We find that SYP132 expression is tightly regulated by auxin and that augmented SYP132 expression reduces the amount of H+-ATPase proteins at the plasma membrane. The physiological consequences of SYP132 overexpression include reduced apoplast acidification and suppressed vegetative growth. Thus, SYP132 plays unexpected and vital roles in auxin-regulated H+-ATPase traffic and associated functions at the plasma membrane., This work was supported by the Royal Society (university research fellowship UF150364 and research grant RG160493 to R.K.). L.X. was supported by a Ph.D. scholarship from the China Scholarship Council. C.G.B. and M.M.M.-B. were supported by University of Glasgow Leadership funds to R.K.

Summary Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1, 2, 3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1, 2, 3, 4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7, 8, 9, 10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface., Graphical Abstract, Highlights • MAKR2 is co-expressed with PIN2 and regulates the pace of root gravitropism • MAKR2 controls PIN2 asymmetric accumulation at the root level during gravitropism • MAKR2 binds to and is a negative regulator of the TMK1 receptor kinase • Auxin antagonizes the MAKR2 inhibition of TMK1 by delocalizing MAKR2 in the cytosol, Marquès-Bueno, Armengot et al. show that the unstructured protein MAKR2 controls the dynamics of the root gravitropic response by acting as a negative regulator of the TMK1 receptor kinase. In addition, the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner.

Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development., A.I.C-D. is a recipient of a BIO2016-78955 grant from the Spanish Ministry of Economy and Competitiveness and a European Research Council, ERC Consolidator Grant (ERC-2015-CoG – 683163). I.B-P. is funded by the FPU15/02822 grant from the Spanish Ministry of Education, Culture and Sport; N.B. by the FI-DGR 2016FI_B 00472 grant from the AGAUR, Generalitat de Catalunya; and A.P-R. by the SEV-2015-0533 from the Severo Ochoa Programme for Centers of Excellence in R&D. M.I. and J.M. acknowledge support from the Spanish Ministry of Economy and Competitiveness and FEDER (EU) through grant FIS2015-66503-C3-3-P, from Ministerio de Ciencia, Innovación y Universidades / Agencia Estatal de Investigación / Fondo Europeo de Desarrollo Regional, Unión Europea through grant PGC2018-101896-B-I00 and from the Generalitat de Catalunya through Grup de Recerca Consolidat 2014 SGR 878 and 2017 SGR 1061. J.M. is funded by the Spanish Ministry of Education through BES-2016-078218. Y.S. and R.C.D. were funded by the Deutsche Forschungsgesellschaft (DFG) (grant STA12/12 1-1). A.I.C.-D.–A.C. collaboration was funded by the European Regional Development Funds and Marie Curie IRSES Project DEANN (PIRSES-GA-2013-612583). CRAG is funded by “Severo Ochoa Programme” from Centers of Excellence in R&D 2016-2019 (SEV-2015-485 0533).

This work was supported by the Royal Society (University Research Fellowship UF150364, Research Grant RG160493; to R.K.) and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S017348/1; to R.K.), the China Scholarship Council (CSC) (to L.X.; a Ph.D. studentship), and the University of Glasgow Leadership funds (to R.K.)

International audience; Rho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a 25 developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 30 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development. One Sentence Summary: 35 Phosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates auxin sensitivity and plant development.

Rho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

Summary The multifunctional protein kinase CK2 is involved in several aspects of the DNA damage response (DDR) in mammals. To gain insight into the role of CK2 in plant genome maintenance, we studied the response to genotoxic agents of an Arabidopsis CK2 dominant-negative mutant (CK2mut plants). CK2mut plants were hypersensitive to a wide range of genotoxins that produce a variety of DNA lesions. However, they were able to activate the DDR after exposure to γ irradiation, as shown by accumulation of phosphorylated histone H2AX and up-regulation of sets of radio-modulated genes. Moreover, functional assays showed that mutant plants quickly repair the DNA damage produced by genotoxins, and that they exhibit preferential use of non-conservative mechanisms, which may explain plant lethality. The chromatin of CK2mut plants was more sensitive to digestion with micrococcal nuclease, suggesting compaction changes that agreed with the transcriptional changes detected for a number of genes involved in chromatin structure. Furthermore, CK2mut plants were prone to transcriptional gene silencing release upon genotoxic stress. Our results suggest that CK2 is required in the maintenance and control of genomic stability and chromatin structure in plants, and that this process affects several functions, including the DNA damage response and DNA repair.

Despite increasing progress in the study of CK2 activity in plants, a clear understanding of its functional role remains elusive. The high pleiotropic nature of the enzyme, the fact that it is absolutely necessary to maintain life, and the existence of multiple isoforms have made it difficult to obtain loss-of-function mutants with which to study the impact of CK2 depletion in the organisms. To avoid all these difficulties, we have used a dominant-negative mutant approach, by constructing a CK2α kinase-inactive subunit (CKA3mut) that was cloned downstream of an inducible promoter. Stably transformed Arabidopsis plants showed that longtime inductions of the transgene were lethal, causing growth and development arrests and ultimately resulting in plant death. However, short-time inductions were not lethal and revealed broad phenotypical changes that uncovered novel functions of CK2 in plants. The high pleiotropy of CK2 was sustained by analysis of global transcript profiles that showed a huge number of genes affected, involved in a wide variety of cellular processes.

Protein kinase CK2 is a pleiotropic Ser/Thr kinase, evolutionary conserved in eukaryotes. Studies performed in different organisms, from yeast to humans, have highlighted the importance of CK2 in cell growth and cell-cycle control. However, the signalling pathways in which CK2 is involved have not been fully identified. In plants, the phytohormone auxin is a major regulator of cell growth. Recent discoveries have demonstrated that differential distribution of within auxin plant tissues is essential for developmental processes, and that this distribution is dependent on polar auxin transport. We report here that a dominant-negative mutant of CK2 (CK2mut) in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. However, CK2mut plants exhibit normal responses to exogenous indole-3-acetic acid (IAA) indicating that they are not affected in the perception of the hormone but upstream in the pathway. We demonstrate that mutant plants are not deficient in IAA but are impaired in its transport. Using genetic and pharmacological tools we show that CK2 activity depletion hinders correct formation of auxin gradients and leads to widespread changes in the expression of auxin-related genes. In particular, members of the auxin efflux carrier family (PINs), and the protein kinase PINOID, both key regulators of auxin fluxes, were misexpressed. PIN4 and PIN7 were also found mislocalized, with accumulation in endosomal bodies. We propose that CK2 functions in the regulation of auxin-signalling pathways, particularly in auxin transport.

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.

International audience; This review highlights our current knowledge on how phosphorylation by protein and phosphatidylinositol kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers.The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism.

International audience; The protein kinase CK2 is a ubiquitous and highly conserved enzyme, the activity of which is vital for eukaryotic cells. We recently demonstrated that CK2 modulates salicylic acid (SA) homeostasis in Arabidopsis thaliana, and that functional interplay between CK2 and SA sustains transcriptional expression of PIN-FORMED (PIN) genes. In this work, we show that CK2 also plays a key role in the transcriptional regulation of PINOID (PID), an AGC protein kinase that modulates the apical/basal localization of auxin-efflux transporters. We show that PID transcription is up-regulated by auxin and by SA and that CK2 is involved in both pathways. On the one hand, CK2 activity is required for proteosome-dependent degradation of AXR3, a member of the AUX/IAA family of auxin transcriptional repressors that must be degraded to activate auxin-responsive gene expression. On the other hand, the role of CK2 in SA homeostasis and, indirectly, in SA-driven PID transcription, was confirmed by using Arabidopsis NahG transgenic plants, which cannot accumulate SA. In conclusion, our results evidence a role for CK2 as a functional link in the negative cross-talk between auxin- and SA-signaling.

International audience; Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. Each membrane compartment has a unique identity and thereby recruits a specific set of proteins 1-3. It has been established for decades that these identities are acquired by the combined presence of specific lipid and protein molecules that act as biochemical landmark on each membrane. For example, small GTPases from the Rab and ADP ribosylation factor (ARF) family as well as Soluble N-ethylmaleimide-sensitive-factor Attachment protein Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsReprints and permissions information is available at www.nature.com/reprints.

13 Pags.- 2 Tabls.- 6 Figs. The definitive version is available at: http://onlinelibrary.wiley.com/doi/10.1111/tpj.12481/abstract, We have previously reported that CK2-defective Arabidopsis thaliana plants (CK2mut plants) were impaired severely in root development and auxin polar transport, and exhibited transcriptional misregulation of auxin-efflux transporters (Plant J., 67, 2011a, 169). In this work we show that CK2mut roots accumulate high levels of salicylic acid (SA) and that the gene that encodes isochorismate synthase (SID2) is overexpressed, strongly suggesting that CK2 activity is required for SA biosynthesis via the shikimate pathway. Moreover, SA activates transcription of CK2-encoding genes and, thus, SA and CK2 appear to be part of an autoregulatory feed-back loop to fine-tune each other's activities. We also show that exogenous SA and constitutive high SA levels in cpr mutants reproduce the CK2mut root phenotypes (decrease of root length and of number of lateral roots), whereas inhibition of CK2 activity in SA-defective and SA-signalling mutants lead to less severe phenotypes, suggesting that the CK2mut root phenotypes are SA-mediated effects. Moreover, exogenous SA mediates transcriptional repression of most of PIN-FORMED (PIN) genes, which is the opposite effect observed in CK2mut roots. These results prompted us to propose a model in which CK2 acts as a link between SA homeostasis and transcriptional regulation of auxin-efflux transporters. We also show that CK2 overexpression in Arabidopsis has neither impact on SA biosynthesis nor on auxin transport, but it improves the Arabidopsis root system. Thus, unlike the outcome in mammals, an excess of CK2 in plant cells does not produce neoplasia, but it might be advantageous for plant fitness. © 2014 John Wiley & Sons Ltd., This work was supported by grants BFU2010-15090 (Ministerio de Educación y Ciencia, Spain) and 2009SGR-795 (Generalitat de Catalunya, Catalunya, Spain). L.A. was recipient of a fellowship from the Ministerio de Educación y Ciencia (Spain). The authors declare to have no conflict of interest.

Protein phosphorylation is one of the most important posttranslational mechanisms regulating polar auxin transport (PAT). The best known example is the phosphorylation carried out by the protein kinase PINOID (PID), which determines PIN polar targeting at the plasma membrane. PID belongs to the family of plant AGC kinases, which play prominent roles in the regulation of PAT and in the cross talk between PAT and the light-signaling pathways. Moreover, kinases belonging to other families, such as protein kinase CK2 or MAP kinases, are now emerging as important regulators of PIN recycling and/or other aspects of PAT, and the phosphoinositide biosynthetic pathway appears to play a significant role on PIN trafficking. On the other hand, identification of protein phosphatases that may reverse the effects of protein kinases is far behind, although rapid progress is being made, providing new insights about the role of protein phosphorylation on PAT regulation.

Protein kinase CK2 is a pleiotropic Ser/Thr kinase, evolutionary conserved in eukaryotes. Studies performed in different organisms, from yeast to humans, have highlighted the importance of CK2 in cell growth and cell-cycle control. However, the signalling pathways in which CK2 is involved have not been fully identified. In plants, the phytohormone auxin is a major regulator of cell growth. Recent discoveries have demonstrated that differential distribution of within auxin plant tissues is essential for developmental processes, and that this distribution is dependent on polar auxin transport. We report here that a dominant-negative mutant of CK2 (CK2mut) in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. However, CK2mut plants exhibit normal responses to exogenous indole-3-acetic acid (IAA) indicating that they are not affected in the perception of the hormone but upstream in the pathway. We demonstrate that mutant plants are not deficient in IAA but are impaired in its transport. Using genetic and pharmacological tools we show that CK2 activity depletion hinders correct formation of auxin gradients and leads to widespread changes in the expression of auxin-related genes. In particular, members of the auxin efflux carrier family (PINs), and the protein kinase PINOID, both key regulators of auxin fluxes, were misexpressed. PIN4 and PIN7 were also found mislocalized, with accumulation in endosomal bodies. We propose that CK2 functions in the regulation of auxin-signalling pathways, particularly in auxin transport.

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.