The bHLH transcription factors TSAR1 and TSAR2 regulate triterpene saponin biosynthesis in Medicago truncatula

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula

CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3BPM in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3BPM-mediated degradation of MYC transcription factors.This work was funded by Spanish Ministry for Science and Innovation Grants BIO2016-77216-R (Ministerio de Economia [MINECO]/Fondos Europeos de Desarrollo Regional [FEDER]) (to R.S.) and BIO2016-80551-R (MINECO/FEDER) (to V.R.). E.C. was the recipient of a Formación de Personal Investigador grant from MINECO (Reference BES-2017-081147). The mass spectrometry instrumentation was funded by the University of Strasbourg (IdEx “Equipement mi-Lourd” 2015) and by “Laboratoires d’Excellence” Grant ANR-10-LABX-0036 (NETRNA)

Characterization of the cytokinin-responsive transcriptome in rice

Abstract Background Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice. Results We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors. Conclusions These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots

PthA4AT, a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to ‘classical’ TALEs.Fil: Roeschlin, Roxana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Uviedo, Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: García, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Molina, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Favaro, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Chiesa, Maria Amalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Tasselli, Sabrina Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Franco Zorrilla, José Manuel. Instituto de Bioquímica y Biología Molecular; ArgentinaFil: Forment, Javier. Universidad Politécnica de Valencia; EspañaFil: Gadea, José. Universidad Politécnica de Valencia; EspañaFil: Marano, María Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Arabidopsis SWC4 Binds DNA and Recruits the SWR1 Complex to Modulate Histone H2A.Z Deposition at Key Regulatory Genes

Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWR1-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWR1-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo-lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and reproductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses transcription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWR1-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.This work was supported by grants BIO2010-15589, BIO2013-43098-R, and BIO2016-77559-R to J.A.J. and M.P., and grant RYC-2013-14689 to P.C. from the Spanish Ministerio de Economia y Competitividad (MINECO/FEDER, EU), and Marie Curie FP7-PEOPLE-2011-IEF grant 298790 to P.C. and J.A.J. from the European Commission. The CBGP is a Severo Ochoa Center of Excellence (SEV-2016-0672). The CNIC is supported by the Spanish Ministerio de Economia, Industria y Competitividad and the Pro CNIC Foundation, and is also a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Chestnut and poplar RAV genes in tree seasonal dormancy

Plants from temperate regions adapt to changing environmental conditions along the year. Trees have evolved mechanisms that allow them to monitor and anticipate the seasons, and cycle between growth and winter dormancy states. Dormancy is initiated by shortening of photoperiod, and afterwards, as a result of a drop in temperature, trees reach a state of endodormancy, the inability of resume growth in response to inductive conditions. Chilling requirement needs to be fulfilled in order to release from endodormancy and gain the ability to resume growth in response to good conditions. The signalling networks that regulate dormancy in perennials are poorly understood. We had previously shown that CsRAV1, a chestnut homolog of Arabidopsis TEM1 and TEM2, induced sylleptic branching in poplar [1]. In this work we characterize the role of chestnut and poplar RAV genes in dormancy. The expression profile of CsRAV1, PtaRAV1 and PtaRAV2 along the year showed that all three genes were induced during winter and maintained high expression levels until early spring. These data suggested that CsRAV1, PatRAV1 and PtaRAV2 were involved in the regulation of winter dormancy in trees. To test this hypothesis we have used over-expressing CsRAV1, and knock-down PtaRAV1 and PtaRAV2 transgenic poplars. The phenology of the transgenic lines will be discussed. It has been reported that Arabidopsis TEM1 binds to the FT promoter. An in silico screening of TEM1 DNA recognition sites in the promoter region of the Populus trichocarpa homologous FT genes revealed that the RAV1 motif was not conserved. Moreover, the over-expression of CsRAV1 in Arabidopsis did not phenocopy the over-expression of AtTEM1 and AtTEM2, suggesting a functional divergence of RAV family members. To gain insight on the molecular function of tree RAV genes, we performed a transcriptomic analysis with RNA from the poplar transgenic lines, and protein-binding microarrays to identify the cis-acting elements for CsRAV1, PtaRAV1 and PtaRAV2. The identification of the binding elements and their occurrence in the genes differentially expressed will be presented. In conclusion, our study reveals a possible function of RAV transcriptional regulators in the control of winter dormancy in trees

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.Peer reviewe

A MYB/ZML complex regulates wound-induced lignin genes in maize

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes.Research in D.C.-R.'s laboratory was supported by a grant from the Spanish Ministry of Science and Education (AGL2011-30545-C02-01), the “Xarxa de Referència de Biotecnologia” (XarBa) from the Autonomous Government of Catalonia, the CONSOLIDER-INGENIO program (CSD2007-00036) from the Spanish Ministry of Science and Innovation, and the SGR programs (SGR2009-GRC703). Research in M.P.'s laboratory was supported by two grants from the Spanish Ministry of Science and Education (BIO2009-13044-C02-01 and BIO2012-31860), the framework of the XarBa, and the SGR programs (SGR2009-GRC626) from the Autonomous Government of Catalonia. Research in R.S.'s laboratory was supported by grants from the Ministry of Science and Innovation to R.S. (BIO2013-44407). M.P. and R.S. received financial support from the CONSOLIDER-INGENIO program (CSD2007-00057-B) from the Spanish Ministerio de Ciencia e Innovación. Research in the W.S. laboratory is supported by grants from the Ministry of Science and Technology and Academia Sinica. Research in phenylpropanoid gene regulation in the laboratories of E.G. and J.G. was supported by a grant from the National Science Foundation (IOS-1125620). I.-C.V.-B. was supported by a Spanish FPI Fellowship (BES-2007-17316). J.E.S.-H. was supported by the Department of Innovation, Universities and Enterprise of the Generalitatde Catalunya, the European Social Fund FI Fellowship (AGAUR: FI-2006, Resolució EDU/3600/2006; FI-2008, Resolució IUE/2658/2007 and BE-DGR2010), and CRAG.Peer reviewe

Consensus document for ultrasound training in the specialty of Nephrology

Grupo de Trabajo en Nefrología Diagnóstica e Intervencionista (GNDI) de la Sociedad Española de Nefrología (SEN).[ES] La ecografía es una herramienta esencial en el manejo del paciente nefrológico que permite el diagnóstico, el seguimiento y la realización de intervencionismo sobre el riñón. La utilidad de los ultrasonidos en Nefrología no se circunscribe exclusivamente al estudio ecográfico del riñón. Mediante ecografía el nefrólogo puede, además, optimizar el manejo de la fístula arteriovenosa para hemodiálisis, medir el riesgo cardiovascular (grosor íntima-media), implantar catéteres centrales para hemodiálisis ecoguiados y ayudar en la colocación de los peritoneales, así como calcular la volemia del paciente mediante ecografía cardiaca básica, ecografía de la vena cava inferior y pulmonar. Desde el Grupo de Trabajo en Nefrología Diagnóstica e Intervencionista (GNDI) de la Sociedad Española de Nefrología (SEN) hemos elaborado este documento de consenso en el que se resumen las principales aplicaciones de la ecografía en Nefrología, incluyendo los requisitos técnicos básicos necesarios, el marco normativo y el nivel de capacitación de los nefrólogos en esta materia. El objetivo de este trabajo es promover la inclusión de la ecografía, tanto diagnóstica como intervencionista, en la práctica clínica habitual del nefrólogo y en la cartera de servicios de Nefrología con la finalidad de ofrecer un manejo diligente, eficiente e integral al paciente nefrológico.[EN] Ultrasound is an essential tool in the management of the nephrological patient allowing the diagnosis, monitoring and performance of kidney intervention. However, the usefulness of ultrasound in the hands of the nephrologist is not limited exclusively to the ultrasound study of the kidney. By ultrasound, the nephrologist can also optimize the management of arteriovenous fistula for hemodialysis, measure cardiovascular risk (mean intimate thickness), implant central catheters for ultrasound-guided HD, as well as the patient's volemia using basic cardiac ultrasound, ultrasound of the cava inferior vein and lungs. From the Working Group on Interventional Nephrology (GNDI) of the Spanish Society of Nephrology (SEN) we have prepared this consensus document that summarizes the main applications of ultrasound to Nephrology, including the necessary basic technical requirements, the framework normative and the level of training of nephrologists in this area. The objective of this work is to promote the inclusion of ultrasound, both diagnostic and interventional, in the usual clinical practice of the nephrologist and in the Nephrology Services portfolio with the final objective of offering diligent, efficient and comprehensive management to the nephrological patient