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12 Pág. Centro de Biotecnología y Genómica de Plantas, Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs., This research has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 260468 to MMC and from the grant S2013-ABI2734 from CAM. In addition, this work has been financially supported by RTI2018-095946-B100 from MICIU and by the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672 [2017–2021]) to the CBGP. In the frame of this latter program, RT was supported with a postdoctoral contract.

12 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP), HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development., This study is supported by the project RTI2018-095946-B-I00 from MICIU to M. Mar Castellano, BIO2017-82209-R and PID2020-113479RB-I00 to Juan C. del Pozo, and by ‘Severo Ochoa Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación of Spain [grant SEV-2016-0672 (2017–2021) to the CBGP]. In the frame of this latter programme, Silvina Mangano and René Toribio were supported with postdoctoral contracts.

14 Pág., Gibberellins (GAs) play important roles in multiple developmental processes and in plant response to the environment. Within the GA pathway, a central regulatory step relies on GA-dependent degradation of the DELLA transcriptional regulators. Nevertheless, the relevance of the stability of other key proteins in this pathway, such as SLY1 and SNE (the F-box proteins involved in DELLA degradation), remains unknown. Here, we take advantage of mutants in the HSP70-HSP90 organizing protein (HOP) co-chaperones and reveal that these proteins contribute to the accumulation of SNE in Arabidopsis. Indeed, HOP proteins, along with HSP90 and HSP70, interact in vivo with SNE, and SNE accumulation is significantly reduced in the hop mutants. Concomitantly, greater accumulation of the DELLA protein RGA is observed in these plants. In agreement with these molecular phenotypes, hop mutants show a hypersensitive response to the GA inhibitor paclobutrazol and display a partial response to the ectopic addition of GA when GA-regulated processes are assayed. These mutants also display different phenotypes associated with alterations in the GA pathway, such as reduced germination rate, delayed bolting, and reduced hypocotyl elongation in response to warm temperatures. Remarkably, ectopic overexpression of SNE reverts the delay in germination and the thermally dependent hypocotyl elongation defect of the hop1 hop2 hop3 mutant, revealing that SNE accumulation is the key aspect of the hop mutant phenotypes. Together, these data reveal a pivotal role for HOP in SNE accumulation and GA signaling., This work is supported by the project RTI2018-095946-B-I00 and PID2021-126956OB-I00 from MICIU and by ‘‘Severo Ochoa Programme for Centres of Excellence in R&D’’ from the Agencia Estatal de Investigacion of Spain (grants SEV-2016-0672 and CEX2020-000999-S to the CBGP). In the frame of this latter program, S.M. was supported with post doctoral contracts. We also acknowledge the grant ‘‘Recualificacio´ n del profesorado universitario’’ from the Ministerio de Universidades to A.M. (UCO), With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S)

Translation is a fundamental step in gene expression that regulates multiple developmental and stress responses. One key step of translation initiation is the association between eIF4E and eIF4G. This process is regulated in different eukaryotes by proteins that bind to eIF4E; however, evidence of eIF4E-interacting proteins able to regulate translation is missing in plants. Here, we report the discovery of CERES, a plant eIF4E-interacting protein. CERES contains an LRR domain and a canonical eIF4E-binding site. Although the CERES–eIF4E complex does not include eIF4G, CERES forms part of cap-binding complexes, interacts with eIF4A, PABP and eIF3, and co-sediments with translation initiation complexes in vivo. Moreover, CERES promotes translation in vitro and general translation in vivo, while it modulates the translation of specific mRNAs related to light and carbohydrate response. These data suggest that CERES is a non-canonical translation initiation factor that modulates translation in plants. Eukaryotic translation initiation factor 4E-interacting proteins able to regulate translation have not yet been reported in plants. A study now characterizes an eIF4E-interacting protein, CERES, as a non-canonical translation initiation factor that modulates translation in plants.

11 Pág. Centro de Biotecnología y Genómica de Plantas, Protein synthesis is a fundamental process for life and, as such, plays a crucial role in the adaptation to energy, developmentaland environmental conditions. For these reasons, and despite the general conservation of the eukaryotic translational machinery, it is not surprising that organisms with different lifestyles have evolved distinct mechanisms of regulation to adapt translation initiation to their intrinsic growth and development. Plants have clear peculiarities compared with other eukaryotes that have also extended to translation control. This review describes the plant-specific mechanisms for regulation of translation initiation, with a focus on those that modulate the eIF4F complexes, central translational regulatory hubs in all eukaryotes, and highlights the latest discoveries on the signaling pathways that regulate their constituents and activity., Our research related to translation is supported by Grants S2013-ABI2734 (CAM, Spain) and RTI2018-095946-Our research related to translation is supported by Grants S2013-ABI2734 (CAM, Spain) and RTI2018-095946-B100 (MICIU, Spain) to M.M.C. and Grants BIO2017-82720-P and RYC2017-22323 (MINECO, Spain) and P18-RT-1218 (Junta de Andalucía, Spain) to C.M.

HOPs (HSP70–HSP90 organizing proteins) are a highly conserved family of HSP70 and HSP90 co-chaperones whose role in assisting the folding of various hormonal receptors has been extensively studied in mammals. In plants, HOPs are mainly associated with stress response, but their potential involvement in hormonal networks remains completely unexplored. In this article we describe that a member of the HOP family, HOP3, is involved in the jasmonic acid (JA) pathway and is linked to plant defense responses not only to pathogens, but also to a generalist herbivore. The JA pathway regulates responses to Botrytis cinerea infection and to Tetranychus urticae feeding; our data demonstrate that the Arabidopsis (Arabidopsis thaliana) hop3-1 mutant shows an increased susceptibility to both. The hop3-1 mutant exhibits reduced sensitivity to JA derivatives in root growth assays and downregulation of different JA-responsive genes in response to methyl jasmonate, further revealing the relevance of HOP3 in the JA pathway. Interestingly, yeast two-hybrid assays and in planta co-immunoprecipitation assays found that HOP3 interacts with COI1, suggesting that COI1 is a target of HOP3. Consistent with this observation, COI1 activity is reduced in the hop3-1 mutant. All these data strongly suggest that, specifically among HOPs, HOP3 plays a relevant role in the JA pathway by regulating COI1 activity in response to JA and, consequently, participating in defense signaling to biotic stresses., One-sentence summary: The co-chaperone protein HOP3 (HSP70-HSP90 ORGANIZING PROTEIN 3) regulates the activity of jasmonic acid co-receptor CORONATINE INSENSITIVE 1 and functions in plant defense.

Translation plays an important role in plant adaptation to different abiotic and biotic stresses, however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2&alpha, phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2&alpha, phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet&ndash, 35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2&alpha, phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.

Translation plays an important role in plant adaptation to different abiotic and biotic stresses; however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2α phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2α phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet–35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2α phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

HSP70-HSP90 organizing protein (HOP) is a well-studied family of cytosolic cochaperones. However, the possible role of HOP during the endoplasmic reticulum (ER) stress response and the identity of its interactors within the ER were not previously addressed in any eukaryote. We have demonstrated that Arabidopsis HOP3, whose function was not studied before, interacts in vivo with cytosolic HSP90 and HSP70, and, unexpectedly, with binding immunoglobulin protein (BiP), a HSP70 ER-resident protein. Although BiP lacks the domain described in other eukaryotes for HOP-HSP70 binding, it interacts with HOP3 through a non-canonical association to its nucleotide binding domain. Consistent with this interaction with BiP, HOP3 is partially localized at the ER. Moreover, HOP3 is induced both at transcript and protein levels by unfolded protein response (UPR) inducer agents by a mechanism dependent on inositol-requiring enzyme 1 (IRE1). Importantly, hop3 loss-of-function mutants show a reduction in pollen germination and a hypersensitive phenotype in the presence of ER stress inducer agents, a phenotype that is reverted by the addition of the chemical chaperone tauroursodeoxycholic acid (TUDCA). All these data demonstrate, for the first time in any eukaryote, a main role of HOP as an important regulator of the ER stress response, a process intimately linked in plants to important specific developmental programs and to environmental stress sensing and response.

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.

36 p.-8 fig.-1 tab.3 fig. supl.+ data sets, The control of pre-mRNA splicing is emerging as an important layer of regulation in plant response to endogenous and external cues. In eukaryotes, pre-mRNA splicing is governed by the activity of a large ribonucleoprotein machinery, the spliceosome, whose protein core is composed of the SM ring and the related SM-like (LSM) 2-8 complex. Recently, the Arabidopsis LSM2-8 complex has been characterized. The role of the plant Sm proteins in pre-mRNA splicing, however, remains largely unknown. Here, we present the functional characterization of SmE1, an Arabidopsis homolog of the SME subunit of the eukaryotic Sm ring. Our results demonstrate that it regulates the spliceosome activity and that this regulation is controlled by the environmental conditions. Indeed, depending on the conditions, SME1 ensures the efficiency of constitutive and alternative splicing of selected pre-mRNAs. Moreover, mis-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that SME1 also guarantees adequate levels of the corresponding functional transcripts. In addition, we show that the selective function of SME1 in ensuring appropriate gene expression patterns through the regulation of specific pre-mRNA splicing is essential for adequate plant development and adaptation to freezing temperatures. These findings reveal that SME1 plays a critical role in plant development and interaction with the environment by providing spliceosome activity specificity., This research was supported by grants BIO2016-79187-R from AEI/FEDER, UE to J.S., BIO2016-77559-R from AEI/FEDER, UE to J.A.J.and M.P.,and RYC-2013-14689 to P.C.

Protein-protein interactions discovered by yeast two-hybrid systems must be confirmed in vivo in a homologous system. In the case of plants, one of the easiest and fastest methods to validate protein interactions in vivo is the transient expression of the proteins in Nicotiana benthamiana leaves followed by coimmunoprecipitation. This method consists of the following steps: growth of the appropriate Agrobacterium tumefaciens cultures, preparation of the infiltration mixtures, infiltration into N. benthamiana leaves, protein extraction and immunoprecipitation. The utilization of epitope tags to immunoprecipitate and detect the proteins of interest is very useful in this procedure. In this chapter we describe a standard protocol to coimmunoprecipitate proteins expressed in N. benthamiana leaves.

In many plant species, an exposure to a sublethal temperature triggers an adaptative response called acclimation. This response involves an extensive molecular reprogramming that allows the plant to further survive to an otherwise lethal increase of temperature. A related response is also launched under an abrupt and lethal heat stress that, in this case, is unable to successfully promote thermotolerance and therefore ends up in plant death. Although these molecular programmes are expected to have common players, the overlapping degree and the specific regulators of each process are currently unknown. We have carried out a high-throughput comparative proteomics analysis during acclimation and during the early stages of the plant response to a severe heat stress that lead Arabidopsis seedlings either to survival or death. This analysis dissects these responses, unravels the common players and identifies the specific proteins associated with these different fates. Thermotolerance assays of mutants in genes with an uncharacterized role in heat stress demonstrate the relevance of this study to uncover both positive and negative heat regulators and pinpoint a pivotal role of JR1 and BAG6 in heat tolerance.

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

Protein-protein interactions discovered by yeast two-hybrid systems must be confirmed in vivo in a homologous system. In the case of plants, one of the easiest and fastest methods to validate protein interactions in vivo is the transient expression of the proteins in Nicotiana benthamiana leaves followed by coimmunoprecipitation. This method consists of the following steps: growth of the appropriate Agrobacterium tumefaciens cultures, preparation of the infiltration mixtures, infiltration into N. benthamiana leaves, protein extraction and immunoprecipitation. The utilization of epitope tags to immunoprecipitate and detect the proteins of interest is very useful in this procedure. In this chapter we describe a standard protocol to coimmunoprecipitate proteins expressed in N. benthamiana leaves.

The authors acknowledge funding by MINECO BIO2015-70483-R to AF, by CAM S2013/ABI-2734 and by ERC GA260468 to MMC, by the Deutsche Forschungsgemeinschaft (DFG, grant TRR175-C05) to DL, by NSF IOS 1444561 and NSF IOS PAPM-EAGER 1650139 to AS, and by Bio4Energy, a Strategic Research Environment appointed by the Swedish government to JH.

HSP70-HSP90 organizing protein (HOP) is a well-studied family of cytosolic cochaperones. However, the possible role of HOP during the endoplasmic reticulum (ER) stress response and the identity of its interactors within the ER were not previously addressed in any eukaryote. We have demonstrated that Arabidopsis HOP3, whose function was not studied before, interacts in vivo with cytosolic HSP90 and HSP70, and, unexpectedly, with binding immunoglobulin protein (BiP), a HSP70 ER-resident protein. Although BiP lacks the domain described in other eukaryotes for HOP-HSP70 binding, it interacts with HOP3 through a non-canonical association to its nucleotide binding domain. Consistent with this interaction with BiP, HOP3 is partially localized at the ER. Moreover, HOP3 is induced both at transcript and protein levels by unfolded protein response (UPR) inducer agents by a mechanism dependent on inositol-requiring enzyme 1 (IRE1). Importantly, hop3 loss-of-function mutants show a reduction in pollen germination and a hypersensitive phenotype in the presence of ER stress inducer agents, a phenotype that is reverted by the addition of the chemical chaperone tauroursodeoxycholic acid (TUDCA). All these data demonstrate, for the first time in any eukaryote, a main role of HOP as an important regulator of the ER stress response, a process intimately linked in plants to important specific developmental programs and to environmental stress sensing and response.

In many plant species, an exposure to a sublethal temperature triggers an adaptative response called acclimation. This response involves an extensive molecular reprogramming that allows the plant to further survive to an otherwise lethal increase of temperature. A related response is also launched under an abrupt and lethal heat stress that, in this case, is unable to successfully promote thermotolerance and therefore ends up in plant death. Although these molecular programmes are expected to have common players, the overlapping degree and the specific regulators of each process are currently unknown. We have carried out a high-throughput comparative proteomics analysis during acclimation and during the early stages of the plant response to a severe heat stress that lead Arabidopsis seedlings either to survival or death. This analysis dissects these responses, unravels the common players and identifies the specific proteins associated with these different fates. Thermotolerance assays of mutants in genes with an uncharacterized role in heat stress demonstrate the relevance of this study to uncover both positive and negative heat regulators and pinpoint a pivotal role of JR1 and BAG6 in heat tolerance.

The Geminiviridae family includes a large number of viruses that infect plants and have a unique geminate virion particle, a single-stranded genome of approximately 2.6-3.0 kb, and replicate through a rolling-circle mechanism. Since they encode for just a few proteins (4-6 depending on the members that belong to four different genera), a rich variety of interactions has evolved between viral proteins and host factors to develop the virus replicative cycle. Among them, we have been particularly interested so far: (i). in the interference with cell cycle regulatory proteins of the retinoblastoma-related (RBR)/E2F pathway and (ii). in the interaction with host DNA replication factors necessary for the assembly of a functional replication complex at the viral origin of DNA replication during the rolling-circle stage. Yeast two-hybrid assays revealed that wheat dwarf virus RepA protein, but nor Rep protein, interacts with plant RBR protein. Interestingly, deletion of the C-terminal domain of Rep confers the truncated protein the ability to interact with RBR, suggesting that this domain may hinder the LXCXE RBR-binding motif. Secondary structure predictions support such a possibility.

The highly conserved Cdc6 protein is required for initiation of eukaryotic DNA replication and, in yeast and Xenopus, for the coupling of DNA replication to mitosis. Herein, we show that human Cdc6 is rapidly destroyed by a p53-independent, proteasome-, and ubiquitin-dependent pathway during early stages of programmed cell death induced by the DNA-damaging drug adozelesin, or by a separate caspase-dependent pathway in cells undergoing apoptosis through an extrinsic pathway induced by tumor necrosis factor-α and cycloheximide. The proteasome-dependent pathway induced by adozelesin is conserved in the budding yeast Saccharomyces cerevisiae. The destruction of Cdc6 may be a primordial programmed death response that uncouples DNA replication from the cell division cycle, which is reinforced in metazoans by the evolution of caspases and p53.

HOPs (heat shock protein 70 (HSP70)-heat shock protein 90 (HSP90) organizing proteins) are a highly conserved family of cytosolic cochaperones. In a recent study we showed that HOP3, a member of the HOP family in Arabidopsis, plays an essential role during endoplasmic reticulum (ER) stress in plants. Interestingly, we also demonstrated that AtHOP3 interacts with binding immunoglobulin protein (BiP), a major ER-resident chaperone. All these data suggest that HOP3 could assist BiP in protein folding in the ER. These findings open the exciting possibility that HOP3, through its role in the alleviation of ER stress, could play an important function during different developmental processes and in response to different biotic and abiotic stresses. © 2017 The Author(s). Published with license by Taylor & Francis Group, LLC

18 p.-10 fig., In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis., This work was supported by the Spanish Secretary of Research, Development, and Innovation (Grants CSD2007-00057, EUI2009-04074, and BIO2010-17545), by grants from the Gordon and Betty Moore Fundation (GMBF3034) and from the National Science Foundation (MCB 1122250) to J.R.E., and by a JAE-DOC contract from the Consejo Superior de Investigaciones Científicas to R.C

The mechanism of initiation of DNA replication in eukaryotic rolling- circle replicons is still poorly understood in molecular terms. Geminiviruses, a family of plant DNA viruses, which use this strategy during part of their replicative cycle, replicate in the nucleus and are amenable to molecular studies. Except for the virally encoded initiator protein (Rep), geminivirus DNA replication relies on cellular factors, likely interfering with cell cycle regulation of the infected cell. Here, we report the identification of three distinct DNA-protein complexes of the DNA replication initiator protein encoded by wheat dwarf geminivirus (WDV) within viral regulatory sequences controlling DNA replication and transcription. We have mapped the WDV Rep binding sites by combining gel-shift assays, electron microscopy and DNase I footprinting. Two of the Rep-DNA complexes (C and the V) are high-affinity complexes, located in the proximity of the two divergent TATA boxes, at 150 and 90 bp, respectively, from the DNA replication initiation site. The third one, the O-complex, is a low-affinity complex, which can assemble under conditions supporting the DNA cleavage reaction. This suggests that it might be responsible for initiation of rolling-circle DNA replication in WDV and other members of the Mastrevirus genus.

[EN] Plants have developed versatile strategies to deal with the great variety of challenging conditions they are exposed to. Among them, the regulation of translation is a common target to finely modulate gene expression both under biotic and abiotic stress situations. Upon environmental challenges, translation is regulated to reduce the consumption of energy and to selectively synthesize proteins involved in the proper establishment of the tolerance response. In the case of viral infections, the situation is more complex, as viruses have evolved unconventional mechanisms to regulate translation in order to ensure the production of the viral encoded proteins using the plant machinery. Although the final purpose is different, in some cases, both plants and viruses share common mechanisms to modulate translation. In others, the mechanisms leading to the control of translation are viral- or stress-specific. In this paper, we review the different mechanisms involved in the regulation of translation initiation under virus infection and under environmental stress in plants. In addition, we describe the main features within the viral RNAs and the cellular mRNAs that promote their selective translation in plants undergoing biotic and abiotic stress situations., This work was supported by the ERC Starting Grant 260468 to M. Mar Castellano.

57 p.-9 fig.-10 fig. supl. Este artículo forma parte de la tesis de Tamara Hernández-Verdeja, que se puede consultar en : http://digital.csic.es/handle/10261/109043, In yeast and animals, Sm-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1-8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8, that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies eleven genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3 and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the eleven LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The complex LSM1-7 is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The complex LSM2-8 is nuclear and is required for pre-mRNA splicing through U6 snRNA stabilization. More important, our results also reveal that these complexes are essential for the correct turnover and splicing of selected developmental-related mRNAs, and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate developmental-related gene expression through the control of mRNA splicing and decay., This work was supported by grants CSD2007-00057, EUI2009-04074 and BIO2010- 17545 from the Spanish Secretary of Research, Development and Innovation.

For years, the study of gene expression regulation of plants in response to stress conditions has been focused mainly on the analysis of transcriptional changes. However, the knowledge on translational regulation is very scarce in these organisms, despite in plants, as in the rest of the eukaryotes, translational regulation has been proven to play a pivotal role in the response to different stresses. Regulation of protein synthesis under abiotic stress was thought to be a conserved process, since, in general, both the translation factors and the translation process are basically similar in eukaryotes. However, this conservation is not so clear in plants as the knowledge of the mechanisms that control translation is very poor. Indeed, some of the basic regulators of translation initiation, well characterised in other systems, are still to be identified in plants. In this paper we will focus on both the regulation of different initiation factors and the mechanisms that cellular mRNAs use to bypass the translational repression established under abiotic stresses. For this purpose, we will review the knowledge from different eukaryotes but paying special attention to the information that has been recently published in plants. © 2012 Alfonso Muoz and M. Mar Castellano.

In the root epidermis of the model plant Arabidopsis thaliana, the hair/non-hair progenitor cells are specified depending on the GL2 homoebox gene expression. The GEM (GL2-expression modulator), a novel component of the pathway that controls both cell division potential and expression of patterning genes by regulating their epigenetic status, is identified. Cell proliferation and cell fate decisions are strictly coupled processes during plant embryogenesis and organogenesis1,2,3,4,5. In the Arabidopsis thaliana root epidermis, expression of the homeobox GLABRA2 (GL2) gene determines hair/non-hair cell fate6,7. This requires signalling of positional information from the underlying cortical layer8,9, complex transcriptional regulation10,11 and a change in chromatin accessibility12. However, the molecular connections among these factors and with cell division are not known. Here we have identified a GL2-expression modulator, GEM, as an interactor of CDT1, a DNA replication protein. GEM also interacts with TTG1 (TRANSPARENT TESTA GLABRA1), a WD40-repeat protein involved in GL2-dependent cell fate decision, and modulates both cell division and GL2 expression. Here we show that GEM participates in the maintenance of the repressor histone H3K9 methylation status of root patterning genes, providing a link between cell division, fate and differentiation during Arabidopsis root development.

We conducted a retrospective analysis of the clinical presentation and the computed tomographic (CT) and ultrasound (US) findings in six episodes of hepatosplenic brucelloma in five patients. In four episodes, the diagnosis was based on clinical history, serology, and characteristic imaging findings. In the other two episodes in the same patient, the diagnosis was suspected after a biopsy was taken. CT was performed in all six cases and US in five. On US, brucellomas were iso- or hypoechogenic with the liver. Hyperechogenic masses were seen in one patient. Brucellomas were very poorly defined and contained small scattered cystic areas. All lesions showed central or marginal gross focal calcification, except multiple lesions in one patient. Contrast-enhanced CT showed predominantly solid masses with irregular borders and fine or thick enhancing trabeculations separating hypodense solid areas and/or small cystic areas. Two patients showed transdiaphragmatic lung invasion by brucelloma, a complication not previously published. In regions where brucellosis is endemic, brucelloma should be included in the differential diagnosis if a hepatic or splenic mass with irregular borders and central or marginal gross focal calcification is detected, and contrast-enhanced CT shows enhancing trabeculations that separate hypodense solid areas and/or small liquid collections.

CDK-cyclin complexes are the universal drivers of cell cycle transitions. Progression through G(1) and transition to S-phase, thereby initiating genome duplication, requires the concerted action of cyclin-dependent kinase (CDK)-cyclin complexes on specific targets. These targets belong to at least two major regulatory networks: the retinoblastoma-related (RBR)/E2F pathway and complexes that are responsible for the initiation of DNA replication. The G(1) phase is central to the integration of signals that regulate both the exit from the cell division cycle to differentiation and the reactivation of cell proliferation. Cellular factors that are involved in these pathways play a role in regulating cell size and number, and organogenesis. As a consequence, they are also involved in determining plant architecture.

Studies on the CDC6 protein, which is crucial to the control of DNA replication in yeast and animal cells, are lacking in plants. We have isolated an Arabidopsis cDNA encoding the AtCDC6 protein and studied its possible connection to the occurrence of developmentally regulated endoreplication cycles. The AtCDC6 gene is expressed maximally in early S-phase, and its promoter contains an E2F consensus site that mediates the binding of a plant E2F/DP complex. Transgenic plants carrying an AtCDC6 promoter-beta-glucuronidase fusion revealed that it is active in proliferating cells and, interestingly, in endoreplicating cells. In particular, the extra endoreplication cycle that occurs in dark-grown hypocotyl cells is associated with upregulation of the AtCDC6 gene. This was corroborated using ctr1 Arabidopsis mutants altered in their endoreplication pattern. The ectopic expression of AtCDC6 in transgenic plants induced endoreplication and produced a change in the somatic ploidy level. AtCDC6 was degraded in a ubiquitin- and proteosome-dependent manner by extracts from proliferating cells, but it was degraded poorly by extracts from dark-grown hypocotyl endoreplicating cells. Our results indicate that endoreplication is associated with expression of the AtCDC6 gene and, most likely, the stability of its product; it also apparently requires activation of the retinoblastoma/E2F/DP pathway. These conclusions may apply to endoreplicating cells in other tissues of the plant and to endoreplicating cells in other eukaryotes.

Heat stress is one of the most prominent and deleterious environmental threats affecting plant growth and development. Upon high temperatures, plants launch specialized gene expression programs that promote stress protection and survival. These programs involve global and specific changes at the transcriptional and translational levels. However, the coordination of these processes and their specific role in the establishment of the heat stress response is not fully elucidated. We have carried out a genome-wide analysis to monitor the changes in the translation efficiency of individual mRNAs of Arabidopsis thaliana seedlings after the exposure to a heat shock stress. Our results demonstrate that translation exerts a wide but dual regulation of gene expression. For the majority of mRNAs, translation is severely repressed, causing a decreased of 50% in the association of the bulk of mRNAs to polysomes. However, some relevant mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of translation. Sequence analyses of the differentially translated mRNAs unravels that some features, such as the 5'UTR G+C content and the cDNA length, may take part in the discrimination mechanisms for mRNA polysome loading. Among the differentially translated genes, master regulators of the stress response stand out, highlighting the main role of translation in the early establishment of the physiological response of plants to elevated temperatures.

Geminivirus DNA replication during the rolling-circle stage depends on the use of a DNA primer, a strategy poorly understood as compared with other eukaryotic viral systems that rely on RNA or protein as primers. Here we have used wheat dwarf virus (WDV) with the aim of elucidating the events leading to recruitment of cell factors at the replication origin. We have identified a novel interaction of WDV Rep, the replication initiation protein, with the large subunit of the wheat replication factor C complex (TmRFC-1). In other systems, the heteropentameric RFC clamp loader complex stimulates loading of DNA polymerase delta to the primer-template. Expression of TmRFC-1 is subjected to cell-cycle regulation, with a peak in early S-phase. We show that WDV Rep stimulates binding of recombinant TmRFC-1 to a model substrate containing a 3'-OH terminus and a WDV Rep-binding site. This was confirmed using cellular fractions enriched for wheat RFC complex, supporting the idea that, in addition to generating a 3'-OH terminus during initiation of DNA replication, WDV Rep could participate in the recruitment of RFC to the newly formed primer. We propose that this pathway may represent an initial event to facilitate the assembly of other replication factors, e.g., PCNA and/or DNA polymerase delta, a model that could also apply to other eukaryotic replicons, such as nanoviruses, circoviruses, and parvoviruses with a similar DNA replication strategy.

Cell division and cell fate decisions are highly regulated processes that need to be coordinated both spatially and temporally for correct plant growth and development. Gaining a deeper molecular and cellular understanding of these links is especially relevant for plant biology since, unlike in animals, formation of new organs is a process that takes place after embryogenesis and continues throughout the entire plant lifespan. The recent identification of a novel factor, GEM, has provided a molecular framework that coordinates cell division to cell fate in the Arabidopsis epidermis. GEM is an inhibitor of cell division through interacting with CDT1, a DNA replication protein. It also inhibits the expression of the homeobox GLABRA2 (GL2) gene that determines the hair/non-hair fate and the pavement/trichome fate in the root and leaf epidermis, respectively. GEM seems to be crucial in controlling the balance of activating/repressing histone modifications at its target promoters.

Recent evidence supports the notion that the eukaryotic polypeptide chain initiation factor (eIF)4G plays a critical bridging role in coordinating other eIF involved in eukaryotic translation initiation. Here we report the isolation and characterization of a 5621-bp cDNA encoding Drosophila eIF4G. The longest ORF predicts a polypeptide of 1666 amino acids with a molecular mass of 183,940 Da and shares 25% amino acid identity with other eIF4G. The 5' untranslated region is 386 nucleotides long and contains seven AUG codons out of frame. The in vitro transcription/translation of the cDNA yielded a major polypeptide, which was specifically immunoprecipitated with an antibody against Drosophila eIF4G. This polypeptide has the same electrophoretic mobility as eIF4G purified from Drosophila melanogaster embryos. A conserved eIF4E-binding motif was found in Drosophila eIF4G. The gene maps at the 102E region of chromosome 4 and spans a genomic region of approximately 16 kb. It was found to contain 15 introns. A single RNA transcript of approximately 5.9 kb was detected by northern blotting of poly(A)-rich RNA prepared from Drosophila adults. The sequence upstream of the transcription initiation site lacks the consensus TATA box, but contains several sequences possibly involved in the regulation of transcription.