Regulación post-transcripcional del desarrollo y la respuesta a estrés abiótico de Arabidopsis thaliana L. mediada por el complejo LSM1-7

59 p.-19 fig.Low temperature is an important determinant in the confi guration of natural plant communities and defi nes the range of distribution and growth of important crops. Some plants, including Arabidopsis , have evolved sophisticated adaptive mechanisms to tolerate low and freezing temperatures. Central to this adaptation is the process of cold acclimation. By means of this process, many plants from temperate regions are able to develop or increase their freezing tolerance in response to low, nonfreezing temperatures. The identifi cation and characterization of factors involved in freezing tolerance are crucial to understand the molecular mechanisms underlying the cold acclimation response and have a potential interest to improve crop tolerance to freezing temperatures. Many genes implicated in cold acclimation have been identifi ed in numerous plant species by using molecular approaches followed by reverse genetic analysis. Remarkably, however, direct genetic analyses have not been conveniently exploited in their capacity for identifying genes with pivotal roles in that adaptive response. In this chapter, we describe a protocol for evaluating the freezing tolerance of both non-acclimated and cold-acclimated Arabidopsis plants. This protocol allows the accurate and simple screening of mutant collections for the identifi cation of novel factors involved in freezing tolerance and cold acclimation.Peer reviewe

Nitric oxide deficiency decreases C-repeat binding factor-dependent and -independent induction of cold acclimation

[EN] Plant tolerance to freezing temperatures is governed by endogenous components and environmental factors. Exposure to low non-freezing temperatures is a key factor in the induction of freezing tolerance in the process called cold acclimation. The role of nitric oxide (NO) in cold acclimation was explored in Arabidopsis using triple nia1nia2noa1-2 mutants that are impaired in the nitrate-dependent and nitrate-independent pathways of NO production, and are thus NO deficient. Here, we demonstrate that cold-induced NO accumulation is required to promote the full cold acclimation response through C-repeat Binding Factor (CBF)-dependent gene expression, as well as the CBF-independent expression of other cold-responsive genes such as Oxidation-Related Zinc Finger 2 (ZF/OZF2). NO deficiency also altered abscisic acid perception and signaling and the cold-induced production of anthocyanins, which are additional factors involved in cold acclimation.We thank Isabel Lopez-Diaz and Esther Carrera for the hormone quantification carried out at the Plant Hormone Quantification Service, IBMCP, Valencia, Spain. This work was supported by grants from MINECO of Spain Government and FEDER EU funds [BIO2014-56067-P, BIO2017-82945-P to JL and BIO2016-79187-R to JS].Costa-Broseta, Á.; Perea-Resa, C.; Castillo, M.; Ruíz, MF.; Salinas, J.; Leon Ramos, J. (2019). Nitric oxide deficiency decreases C-repeat binding factor-dependent and -independent induction of cold acclimation. Journal of Experimental Botany. 70(12):3283-3296. https://doi.org/10.1093/jxb/erz115S328332967012Adams, S., & Carré, I. A. (2011). Downstream of the plant circadian clock: output pathways for the control of physiology and development. 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Journal of Experimental Botany, 65(4), 907-921. doi:10.1093/jxb/ert454Li, D., Li, Y., Zhang, L., Wang, X., Zhao, Z., Tao, Z., … Yang, Y. (2014). Arabidopsis ABA Receptor RCAR1/PYL9 Interacts with an R2R3-Type MYB Transcription Factor, AtMYB44. International Journal of Molecular Sciences, 15(5), 8473-8490. doi:10.3390/ijms15058473Lozano-Juste, J., Colom-Moreno, R., & León, J. (2011). In vivo protein tyrosine nitration in Arabidopsis thaliana. Journal of Experimental Botany, 62(10), 3501-3517. doi:10.1093/jxb/err042Lozano-Juste, J., & León, J. (2009). Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis. Plant Physiology, 152(2), 891-903. doi:10.1104/pp.109.148023Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi, A., Yabuta, Y., & Shigeoka, S. (2009). Arabidopsis NAC Transcription Factor, ANAC078, Regulates Flavonoid Biosynthesis under High-light. 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Prefoldins contribute to maintaining the levels of the spliceosome LSM2–8 complex through Hsp90 in Arabidopsis

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2–8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2–8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2–8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.Fil: Esteve Bruna, David. Universidad Politécnica de Valencia; EspañaFil: Carrasco López, Cristian. Consejo Superior de Investigaciones Científicas; EspañaFil: Blanco Touriñán, Noel. Universidad Politécnica de Valencia; EspañaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Calleja Cabrera, Julián. Universidad Politécnica de Valencia; EspañaFil: Perea Resa, Carlos. Consejo Superior de Investigaciones Científicas; EspañaFil: Úrbez, Cristina. Universidad Politécnica de Valencia; EspañaFil: Carrasco, Pedro. Universidad Politécnica de Valencia; EspañaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blázquez, Miguel A.. Universidad Politécnica de Valencia; EspañaFil: Salinas, Julio. Consejo Superior de Investigaciones Científicas; EspañaFil: Alabadí, David. Universidad Politécnica de Valencia; Españ

Redox feedback regulation of ANAC089 signaling alters seed germination and stress response

The interplay between the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO) regulates seed germination and post-germinative seedling growth. We show that GAP1 (germination in ABA and cPTIO 1) encodes the transcription factor ANAC089 with a critical membrane-bound domain and extranuclear localization. ANAC089 mutants lacking the membrane-tethered domain display insensitivity to ABA, salt, and osmotic and cold stresses, revealing a repressor function. Whole-genome transcriptional profiling and DNA-binding specificity reveals that ANAC089 regulates ABA- and redox-related genes. ANAC089 truncated mutants exhibit higher NO and lower ROS and ABA endogenous levels, alongside an altered thiol and disulfide homeostasis. Consistently, translocation of ANAC089 to the nucleus is directed by changes in cellular redox status after treatments with NO scavengers and redox-related compounds. Our results reveal ANAC089 to be a master regulator modulating redox homeostasis and NO levels, able to repress ABA synthesis and signaling during Arabidopsis seed germination and abiotic stress.Peer reviewe

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress

Redox feedback regulation of ANAC089 signaling alters seed germination and stress response

21 p.-4 fig.-2 tab. 1 graph. abst.The interplay between the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO) regulates seed germination and post-germinative seedling growth. We show that GAP1 (germination in ABA and cPTIO 1) encodes the transcription factor ANAC089 with a critical membrane-bound domain and extranuclear localization. ANAC089 mutants lacking the membrane-tethered domain display insensitivity to ABA,salt, and osmotic and cold stresses, revealing a repressor function. Whole-genome transcriptional profiling and DNA-binding specificity reveals that ANAC089 regulates ABA- and redox-related genes. ANAC089 truncated mutants exhibit higher NO and lower ROS and ABA endogenous levels, alongside an altered thiol and disulfide homeostasis. Consistently, translocation of ANAC089 to the nucleus is directed by changes in cellular redox status after treatments with NO scavengers and redox-related compounds. Our results reveal ANAC089 to be a master regulator modulating redox homeostasis and NO levels, able to repress ABA synthesis and signaling during Arabidopsis seed germination and abiotic stress.Wethank the Spanish networks BIO2015-68957-REDT and RED2018-102397-T for stimulating discussions, as well as Dr. José M. Carrasco and Dr. Pablo Vera (IBMCP-CSIC) for help with the protein-expression experiments of the PBM. This work was financed by grants EcoSeed Impacts of Environmental Conditions on Seed Quality ‘‘EcoSeed-311840’’ ERC.KBBE.2012.1.1-01;BIO2017-85758-R and CSD2007-00057 (TRANSPLANTA) from the Ministerio de Ciencia, Innovación y Universidades (MICIU) (Spain); SA313P18 and SA137P20 from Junta de Castilla y León; Escalera de Excelencia CLU-2018-04 co-funded by the P.O. FEDER of Castilla y León 2014–2020 Spain (to O.L.); and the PhD and University Teacher Training Fellowship, Spanish Ministry of Science and Education (to P.A.).Peer reviewe

Identification of Arabidopsis mutants with altered freezing tolerance

18 p.-3 fig.Low temperature is an important determinant in the configuration of natural plant communities and defines the range of distribution and growth of important crops. Some plants, including Arabidopsis thaliana, have evolved sophisticated adaptive mechanisms to tolerate freezing temperatures. Central to this adaptation is the process of cold acclimation. By means of this process, many plants from temperate regions are able to develop or increase their freezing tolerance in response to low, nonfreezing temperatures. The identification and characterization of factors involved in freezing tolerance is crucial to understand the molecular mechanisms underlying the cold acclimation response and has a potential interest to improve crop tolerance to freezing temperatures. Many genes implicated in cold acclimation have been identified in numerous plant species by using molecular approaches followed by reverse genetic analysis. Remarkably, however, direct genetic analyses have not been conveniently exploited in their capacity for identifying genes with pivotal roles in that adaptive response. In this chapter, we describe a protocol for evaluating the freezing tolerance of both nonacclimated and cold acclimated Arabidopsis plants. This protocol allows for the accurate and simple screening of mutant collections for the identification of novel factors involved in freezing tolerance and cold acclimation.Peer reviewe

Prefoldins negatively regulate cold acclimation in Arabidopsis thaliana by promoting nuclear proteasome-mediated HY5 degradation

14 p.-8 fig.The process of cold acclimation is an important adaptive response whereby many plants from temperate regions increase their freezing tolerance after being exposed to low non-freezing temperatures. The correct development of this response relies on proper accumulation of a number of transcription factors that regulate expression patterns of cold-responsive genes. Multiple studies have revealed a variety of molecular mechanisms involved in promoting the accumulation of these transcription factors. Interestingly, however, the mechanisms implicated in controlling such accumulation to ensure their adequate levels remain largely unknown. In this work, we demonstrate that prefoldins (PFDs) control the levels of HY5, an Arabidopsis transcription factor with a key role in cold acclimation by activating anthocyanin biosynthesis, in response to low temperature. Our results show that, under cold conditions, PFDs accumulate into the nucleus through a DELLA-dependent mechanism, where they interact with HY5, triggering its ubiquitination and subsequent degradation. The degradation of HY5 would result, in turn, in anthocyanin biosynthesis attenuation, ensuring the accurate development of cold acclimation. These findings uncover an unanticipated nuclear function for PFDs in plant responses to abiotic stresses.This research was funded by grants BIO2013-47788-R from MINECO and BIO2016-79187-R from AEI/FEDER, UE to J.S and BIO2013-46539-R from MINECO and BIO2016-80551-R from AEI/FEDER, UE to V.R.Peer reviewe

LSM proteins provide accurate splicing and decay of selected transcripts to ensure normal arabidopsis development

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/10261/109043In 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.Peer reviewe