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

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes

Caracterización funcional del factor de transcripción ZmZIM91 de maíz

Trabajo realizado en el Departamento de Génetica Molecular del Centre de Recerca Agrigenòmica (CRAG-CSIC).[ES] La subunidad reguladora β1 de la proteína quinasa de CK2 de maíz fue usada como anzuelo para realizar un cribado mediante doble híbrido de una librería de cDNA, construida a partir de mRNA de hoja de maíz joven sometida a tres horas de deshidratación. Entre las proteínas que interaccionaron con CK2, fue identificado un factor de transcripción putativo llamado ZmZIM91 (Tipo GATA/Zinc Finger). La proteína ZmZIM91 no había sido anteriormente caracterizada en maíz y la información conocida de sus homólogos en Arabidopsis era muy escasa y no proporcionaban datos acerca de las funciones específicas de este grupo de proteínas. Estudios filogenéticos permitieron demostrar que ZmZIM91 pertenecía a la familia TIFY, más concretamente a la subfamilia ZML, a esta gran familia también pertenecen las proteínas JAZ, las cuales han sido muy estudiadas tanto en Arabidopsis como en otras especies. Con el fin de realizar una caracterización molecular de ZmZIM91, en primer lugar, se estableció que esta proteína era sustrato de la proteína quinasa CK2 de maíz, la cual regula a este factor de transcripción a través de fosforilación tanto bajo tratamientos de sequía como de Metil Jasmonato (MeJA). Por otra parte, fueron llevados a cabo experimentos de pull-down, doble híbrido y complementación Bimolecular, los cuales arrojaron resultados como la interacción de ZmZIM91 con la subunidad reguladora β1 de la proteína quinasa CK2, la dimerización de dicho factor y la interacción con las proteína JAZ, sin embargo se estableció que a diferencia de los JAZ, ZmZIM91 no puede interaccionar con COI1. Por otra parte, se realizaron experimentos de western de hojas de maíz silvestres usando el anticuerpo contra la proteína endógena ZmZIM91 y de sobreexpresión en protoplastos de maíz con aplicación de la hormona MeJA, al igual que MeJA y MG132, donde se observó que ZmZIM91 era degradada en presencia de MeJA a una hora y que su degradación era a través de la vía del proteasoma 26S. Mediante un ensayo de Protein-Array, se logró establecer que ZmZIM91 se podía unir a ADN directamente a través de los motivos GATA y GATC. Ensayos de Inmunoprecipitación de la Cromatina y secuenciación masiva paralela (ChIP-Seq) en planta, permitieron determinar genes diana del factor de transcripción ZmZIM91, siendo dos de ellos el maize caffeic acid O-methyltransferase (COMT) implicado en la biosíntesis de lignina y el gen de la DFR (A1) involucrado en la biosíntesis de los flavonoides, estos resultados fueron confirmados a través de ChIP-QPCR, donde fueron detectados como altamente enriquecidos, además mediante un experimento de expresión transciente en protoplastos de maíz, se pudo determinar que el factor de transcripción ZmZIM91 es un represor de COMT y A1, sugiriendo que ZmZIM91 tiene un papel extendido en la ruta de los fenilpropanoides y que además es un regulador negativo de dicha ruta. Experimentos de inmunoprecipitación de la Cromatina (ChIP) con aplicación de tratamiento con MeJA a una hora, confirmaron que en presencia de esta hormona, ZmZIM91 pierde capacidad de unión a los promotores de COMT y A1. Por otra parte, a través de ensayos de complementación Bimolecular se estableció la interacción del factor de transcripción ZmZIM91 con los factores ZmMYB31 y ZmMYB42 implicados en la regulación negativa de genes de la ruta de los fenilpropanoides, con los cuales podría estar actuando como un módulo regulador sobre promotores de genes como ZmCOMT y ZmA1.[EN] The regulatory subunit of protein kinase CK2β1 was used as a bait to perform a yeast two-hybrid screening against a cDNA library of drought-stressed maize leaves. Between the interacting partners a putative transcription factor named ZmZIM91 (GATA/Zinc Finger type) was identified. The protein ZmZIM91 has not been previously characterized in maize and there was little information about their homologous in Arabidopsis thus, no fuctional data regarding this group of proteins was available. Sequence analysis and phylogenetic studies show that ZmZIM91 belong to the TIFY family, more specifically at the ZML subfamily. JAZ proteins, which have been widely characterized in Arabidopsis and in other species, also belong to the TIFY family. To make a molecular characterization of ZmZIM91, first of all, it was demonstrated that this protein was an in vitro substrate of the maize protein kinase CK2. In addition, in vivo gel kinase assays suggest that phosphorylation regulates this protein under different treatments as drought or methyl jasmonate (MeJA). Furthermore, were carried out a set of experiments as pull-down, yeast two-hybrid assay and Bimolecular fluorescence complementation, which yielded results as the interaction between ZmZIM91 with the regulatory subunits of the protein kinase ZmCK2, the dimerization of ZmZIM91 and the interaction with JAZ protein, however, it was established that unlike JAZ, ZmZIM91 cannot interact with the E3 ubiquitin ligase COI1. Furthermore, were performed western blot experiments using wild-type maize leaves and the endogenous antibody against ZmZIM91 (produced in this work), also assays of overexpression in maize protoplasts in both using the MeJA hormone, as MeJA and MG132, where it was observed that ZmZIM91 was degraded in the presence of MeJA in one hour and its degradation it is probably through the 26S proteasome pathway. Using a Protein-Array assay, it was established that ZmZIM91 could bind to DNA directly through GATA and GATC motifs. Finally, experiments of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq), allowed to determine target genes of the transcription factor ZmZIM91. Between the targets identified, one correspond to the caffeic acid O-methyltransferase (COMT) gene, involved in the biosynthesis of lignin and another is the DFR gene (A1) involved in the biosynthesis of flavonoids. These results were confirmed by ChIP-QPCR, and by transient expression experiments in maize protoplasts where they were identified as highly enriched, It was determined that the ZmZIM91 transcription factor is a repressor of COMT and A1 genes, suggesting a regulatory role of ZmZIM91 in the phenylpropanoid pathway and also as a negative regulator of this pathway. Experiments Chromatin immunoprecipitation (ChIP) with MeJA treatment application at one hour, confirmed that in the presence of this hormone ZmZIM91 loses binding capacity to the COMT and A1 promoters. Moreover, through Bimolecular complementation assays was established the interaction between the transcription factor ZmZIM91 and the ZmMYB31 and ZmMYB42 factors involved in the negative regulation of genes of the phenylpropanoid pathway, which may be acting as a regulatory module to regulate genes such as COMT.Ministerio de Ciencia e Innovación: Beca FPI.Peer reviewe

Specific characteristics of CK2β regulatory subunits in plants

In all eukaryotes, the typical CK2 holoenzyme is an heterotetramer composed of two catalytic (CK2α and CK2α′) and two regulatory (CK2β) subunits. One of the distinctive traits of plant CK2 is that they present a greater number of genes encoding for CK2α/β subunits than animals or yeasts, for instance, in Arabidopsis and maize both CK2α/β subunits belong to multigenic families composed by up to four genes. Here, we conducted a genome-wide survey examining 34 different plant genomes in order to investigate if the multigenic property of CK2β genes is a common feature through the entire plant kingdom. Also, at the level of structure, the plant CK2β regulatory subunits present distinctive features as (i) they lack about 20 aminoacids in the C-terminal domain, (ii) they present a specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain, and (iii) the acidic loop region is poorly conserved at the aminoacid level. Since there is no data about CK2β or holoenzyme structure in plants, in this study, we use human CK2β as a template to predict a structure for Zea mays CK2β1 by homology modeling and we discuss about possible structural changes in the acidic loop region that could affect the enzyme regulation.M.R. was financed by I3P-CSIC2006 and CONSOLIDER (CSD2007-00057) from MICINN (Spain), I.C. V–B. by predoctoral fellowship FPI2007 from MICINN (Spain) and L. C–P. by Juan de la Cierva Programme, MICINN (Spain). This work was supported by grant BIO2009-13044-CO2-01 from MICINN (Spain).Peer reviewe

Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads

Co-immunoprecipitation (Co-IP) is a widely used and powerful approach for studying protein-protein interactions in vivo. Here, we describe a protocol for antibody purification and immobilization followed by immunoprecipitation from plant tissue extracts using magnetic beads. The protocol has been used to detect regulators in the Zea mays phenylpropanoid pathway. The protocol is amenable to a variety of downstream assays, including western blotting and mass spectrometry. For complete details on the use and execution of this protocol, please refer to Vélez-Bermúdez et al. (2015).David Caparrós-Ruiz was supported by the Spanish project AGL2014-58126-R funded by the Ministerio de Economía y Competitividad /AEI and European Regional Development Fund (FEDER). This work was also supported by the SGR programs (SGR2009-GRC703 and 2017SGR710) from the Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya and by the CERCA Programme/Generalitat de Catalunya. We also acknowledge financial support from “Severo Ochoa Programme for Centres of Excellence in R&D” SEV-2015-0533 and CEX2019-000902-S both funded by MCIN/AEI/10.13039/501100011033. I.C.V.-B. was supported by a Ministry of Science and Technology Fellowship (104-2811-12-001-025) and Academia Sinica Postdoctoral Fellowship (L004). J.E.S.-H. was supported by Academia Sinica Postdoctoral Fellowship (L004). Work in the Schmidt laboratory is supported by Academia Sinica and the Ministry of Science and Technology, Taiwan.Peer reviewe

Role of Plant-Specific N-Terminal domain of maize CK2β1 subunit in CK2β functions and holoenzyme regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes

Novel CK2α and CK2β subunits in maize reveal functional diversification in subcellular localization and interaction capacity

In plants, CK2α/β subunits are encoded by multigenic families. They assemble as heterotetrameric holoenzymes or remain as individual subunits and are usually located in distinct cell compartments. Here we revise the number of maize CK2α/β genes, bringing them up to a total of eight (four CK2α catalytic and four CK2β regulatory subunits). We characterize CK2β4, which presents nuclear localization and interacts with CK2α1, CK2α3, CK2β1, and CK2β3. We also describe two CK2α isoforms (CK2α2 and CK2α4) containing N-terminal extensions that correspond to putative cTPs (chloroplast transit peptides). These cTPs are functional and responsible for the subcellular localization of CK2α2 and CK2α4 in chloroplasts. Phylogenetic analysis of the CK2α gene family, further supported by the gene structure and architecture of conserved protein domains, reveals the evolutionary expansion and diversification of this family. The subcellular localization of all four CK2α isoforms was found to be altered when were co-expressed with CK2β, thereby pointing to the latter as regulators of CK2α localization.ICV-B was financed by predoctoral fellowship FPI2007 from MICINN (Spain), TL by JAE CSIC pre-doctoral fellowship and MR by CONSOLIDER-INGENIO 2010 MEC (CSD2007-00057). This work was also supported by grant BIO2012-31860.Peer reviewe

Memorias. Encuentro de Experiencias en Inventarios y Monitoreo Biológico

Las discusiones temáticas alrededor de la consolidación del Inventario Nacional de Biodiversidad para Colombia y la Red de Monitoreo de Biodiversidad como una estrategia de largo plazo, sin duda temas complejos que requerirán de grandes esfuerzos, coordinación y generosidad institucional y personal, los podrá apreciar el lector a lo largo del presente documento, esperando que pueda entender también la importancia que tienen los resultados y la agenda propuesta si en el futuro queremos tomar decisiones con bases científicas