The ABCF3 Gene of Arabidopsis Is Functionally Linked with GCN1 but Not with GCN2 During Stress and Development

[EN] One of the main mechanisms regulating translation is the one based on the phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2 alpha) by the general control non-repressive 2 (GCN2) protein kinase. In yeast, this kinase binds to two scaffold proteins (GCN1 and GCN20), facilitating its activation on translating ribosomes. The homology of the three proteins exists in Arabidopsis. In this species, whereas the kinase is activated under several stress situations, the involvement of the scaffold proteins in those processes is controversial, and a new role for GCN1 in translation, independent of the phosphorylation of eIF2 alpha, has been proposed. Arabidopsis presents five genes with homology to GCN20 (ABCF1 to 5) in its genome. We show here that any of these five genes is needed for eIF2 alpha phosphorylation. Furthermore, plant phenotypes under abiotic stresses and chloroplast development suggest that ABCF3 is functionally linked with GCN1, but not with GCN2. Finally, gcn1 and abcf3 mutants share similar transcriptional reprogramming, affecting photosynthesis and stress responses. The common downregulation of regulators of the flagellin receptor FLS2 in both mutants suggest that the observed defect in pathogen-associated molecular pattern (PAMP)-induced stomatal closure of these two mutants could be mediated by these proteins.This work was funded by the Spanish Ministerio de Ciencia e Innovacion (MICINN), reference BFU2011-22526. Vigya Kesari thanks the EC for an Erasmus Mundus postdoctoral fellowship.Faus, I.; Niñoles Rodenes, R.; Kesari, V.; Gadea Vacas, J. (2021). The ABCF3 Gene of Arabidopsis Is Functionally Linked with GCN1 but Not with GCN2 During Stress and Development. Plant Molecular Biology Reporter. 39(4):663-672. https://doi.org/10.1007/s11105-021-01283-wS66367239

Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection

[EN] The development of a high-performance assay readout using integrated detectors is a current challenge in the implementation of DNA tests in diagnostic laboratories, particularly for supporting pharmacogenetic tests. A method for allelic discrimination, associated with single nucleotide polymorphisms (SNPs), is presented. Genomic DNA is extracted from blood and buccal swab samples. The procedure comprises fast multiplex ligation-dependent probe amplification, PCR amplification using universal primers and subsequent barcode hybridization. In this last step, each product is recognized by the specific probes immobilized on the surface of an optical disc. Assay results can be obtained with a disc reader. The optical sensing method in a DNA microarray format was optimized and evaluated for the simultaneous identification of 28 polymorphisms associated with psychiatric pharmacogenomics. The target biomarkers were located in the genes related to drug-metabolizing enzymes and drug transporters. The multiplexing capability and assay selectivity strongly depended on correct design (ligation probes, tails and barcodes). The discriminant analysis of reader outputs (spot intensities) led to patients being classified into different allelic populations. The obtained assignations correlated properly with the results provided by the reference technique (bead arrays), and the assay ended in an 8-fold shorter time using affordable equipment. The combination of a highly selective genotyping reaction as array-MLPA and the compact disc technology provides a reliable point-of-care approach. This genotyping tool is useful for the selection of personalized drug therapies in decentralized clinical laboratories.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 Project) through the FEDER funds and the Spanish Ministry of Economy and Competitiveness (the MINECO CTQ2016-75749- R Project)Tortajada-Genaro, LA.; Niñoles Rodenes, R.; Mena-Mollá, S.; Maquieira Catala, A. (2019). Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection. The Analyst. 144:707-715. https://doi.org/10.1039/C8AN01830HS70771514

Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2alpha phosphorylation

[EN] One of the main mechanisms blocking translation after stress situations is mediated by phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2), performed in Arabidopsis by the protein kinase GCN2 which interacts and is activated by ILITHYIA(ILA). ILA is involved in plant immunity and its mutant lines present phenotypes not shared by the gcn2 mutants. The functional link between these two genes remains elusive in plants. In this study, we show that, although both ILA and GCN2 genes are necessary to mediate eIF2 alpha phosphorylation upon treatments with the aromatic amino acid biosynthesis inhibitor glyphosate, their mutants develop distinct root and chloroplast phenotypes. Electron microscopy experiments reveal that ila mutants, but not gcn2, are affected in chloroplast biogenesis, explaining the macroscopic phenotype previously observed for these mutants. ila3 mutants present a complex transcriptional reprogramming affecting defense responses, photosynthesis and protein folding, among others. Double mutant analyses suggest that ILA has a distinct function which is independent of GCN2 and eIF2 alpha phosphorylation. These results suggest that these two genes may have common but also distinct functions in Arabidopsis.Microarray experiments were done in the Genomics Facility of the IBMCP. MTH was supported by the Austrian Science Found (FWF) projectF03707. This work has been supported by the Spanish Ministry for Science and Education (Plan Nacional 2008-2011).Faus, I.; Niñoles Rodenes, R.; Kesari, V.; Llabata, P.; Tam, E.; Nebauer, SG.; Santiago, J.... (2018). Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2alpha phosphorylation. Journal of Plant Physiology. 224:173-182. https://doi.org/10.1016/j.jplph.2018.04.003S17318222

Comparative analysis of wild-type accessions reveals novel determinants of Arabidopsis seed longevity

Understanding the genetic factors involved in seed longevity is of paramount importance in agricultural and ecological contexts. The polygenic nature of this trait suggests that many of them remain undiscovered. Here, we exploited the contrasting seed longevity found amongst Arabidopsis thaliana accessions to further understand this phenomenon. Concentrations of glutathione were higher in longer-lived than shorter-lived accessions, supporting that redox poise plays a prominent role in seed longevity. However, high seed permeability, normally associated with shorter longevity, is also present in long-lived accessions. Dry seed transcriptome analysis indicated that the contribution to longevity of stored messenger RNA (mRNAs) is complex, including mainly accession-specific mechanisms. The detrimental effect on longevity caused by other factors may be counterbalanced by higher levels of specific mRNAs stored in dry seeds, for instance those of heat-shock proteins. Indeed, loss-of-function mutant analysis demonstrated that heat-shock factors HSF1A and 1B contributed to longevity. Furthermore, mutants of the stress-granule zinc-finger protein TZF9 or the spliceosome subunits MOS4 or MAC3A/MAC3B, extended seed longevity, positioning RNA as a novel player in the regulation of seed viability. mRNAs of proteins with putative relevance to longevity were also abundant in shorter-lived accessions, reinforcing the idea that resistance to ageing is determined by multiple factors.Peer reviewe

Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

[EN] Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H+-ATPase) is indirect: over-expression of ROF2 activates K+ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H+ pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.This work was supported by grants BFU2008-00604 from the Ministerio de Ciencia e Innovacion (Madrid, Spain) and PROMETEO/2010/ 038 of the 'Conselleria de Educacion' (Valencia, Spain). We thank Dr Eugenio Grau (Sequencing Service, Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for sequencing of the various genes, and Dr Vicente Fornes (Instituto de Tecnologia Quimica, Valencia, Spain) for assistance with atomic absorption spectrophotometry. None of the authors has a conflict of interest to declare.Bissoli, G.; Niñoles Rodenes, R.; Fresquet Corrales, S.; Palombieri, S.; Bueso Ródenas, E.; Rubio, L.; Garcia-Sanchez, MJ.... (2012). Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. Plant Journal. 70(4):704-716. https://doi.org/10.1111/j.1365-313X.2012.04921.xS70471670

Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics

[EN] Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant,Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1,SSLEA,SSTPR,DHAR1,CYP86A8,MYB47andSPCH) and five negative ones (RBOHD,RBOHE,RBOHF,KNAT7andSEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.Ministerio de Ciencia, Innovacion y Universidades, Grant/Award Number: BIO2017-88898-PRenard, J.; Niñoles Rodenes, R.; Martínez-Almonacid, I.; Gayubas, B.; Mateos-Fernández, R.; Bissoli, G.; Bueso Rodenas, E.... (2020). Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics. Plant Cell & Environment. 43(10):2523-2539. https://doi.org/10.1111/pce.13822S25232539431

PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis

[EN] Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1-2D, a gain-of-function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild-type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide-soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevityRenard, J.; Martínez-Almonacid, I.; Sonntag, A.; Molina, I.; Moya-Cuevas, J.; Bissoli, G.; Muñoz-Bertomeu, J.... (2020). PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis. Plant Cell & Environment. 43(2):315-326. https://doi.org/10.1111/pce.13656S315326432Almagro, L., Gómez Ros, L. V., Belchi-Navarro, S., Bru, R., Ros Barceló, A., & Pedreño, M. A. (2008). Class III peroxidases in plant defence reactions. 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A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin. Plant Physiology, 122(4), 1119-1128. doi:10.1104/pp.122.4.1119Rains, M. K., Gardiyehewa de Silva, N. D., & Molina, I. (2017). Reconstructing the suberin pathway in poplar by chemical and transcriptomic analysis of bark tissues. Tree Physiology, 38(3), 340-361. doi:10.1093/treephys/tpx060Russell, W. R., Burkitt, M. J., Scobbie, L., & Chesson, A. (2005). EPR Investigation into the Effects of Substrate Structure on Peroxidase-Catalyzed Phenylpropanoid Oxidation. Biomacromolecules, 7(1), 268-273. doi:10.1021/bm050636oSano, N., Rajjou, L., North, H. M., Debeaujon, I., Marion-Poll, A., & Seo, M. (2015). Staying Alive: Molecular Aspects of Seed Longevity. Plant and Cell Physiology, 57(4), 660-674. doi:10.1093/pcp/pcv186Shigeto, J., Itoh, Y., Hirao, S., Ohira, K., Fujita, K., & Tsutsumi, Y. (2015). Simultaneously disrupting AtPrx2 , AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem. 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Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder

Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance.This research has been funded through projects FEDER MINECO INNPACTO IPT-2011-1132-010000, CTQ/2013/45875R, and PrometeoII/2014/040 (GVA).Tortajada-Genaro, LA.; Mena-Mollá, S.; Niñoles Rodenes, R.; Puigmule, M.; Viladevall, L.; Maquieira Catala, Á. (2016). Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Analytical and Bioanalytical Chemistry. 408(9):2339-2345. https://doi.org/10.1007/s00216-016-9332-3S233923454089Cortese S. The neurobiology and genetics of Attention-Deficit/Hyperactivity Disorder (ADHD): what every clinician should know. Eur J Paediatr Neurol. 2012;16:422–33.Contini V, Rovaris DL, Victor MM, Grevet EH, Rohde LA, Bau CH. Pharmacogenetics of response to methylphenidate in adult patients with attention-deficit/hyperactivity disorder (ADHD): a systematic review. Eur Neuropsychopharmacol. 2013;23:555–60.Gardiner SJ, Begg EJ. 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Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva. Clin Chim Acta. 2014;429:198–205

A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions

[EN] The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H+ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H+ efflux (H+ -ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+ -ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K+ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+ -ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+ -ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H+ -ATPase while the guard-cell kinase SnRK2.6/OST1 did not.This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish 'Ministerio de Economia y Competitividad', Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the 'Generalitat Valenciana', Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish 'Consejo Superior de Investigaciones Cientificas', Madrid, Spain. We thank Dr Toshinori Kinoshita (Nagoya University, Nagoya, Japan) for the rabbit antibody against the last 9 aa of AHA2 H+-ATPase with the penultimate Thr947 phosphorylated. We also thank the Proteomics Facility of the 'Centro Nacional de Biotecnologia', Madrid, Spain, for the attempts to identify the phosphorylation site of the H+-ATPase.Planes Ferrer, MD.; Niñoles Rodenes, R.; Rubio, L.; Bissoli, G.; Bueso Ródenas, E.; Garcia-Sanchez, MJ.; Alejandro Martínez, S.... (2015). A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions. Journal of Experimental Botany. 66(3):813-825. https://doi.org/10.1093/jxb/eru442S81382566

Biología molecular de la regulación de la homeostasis de pH en Arabidopsis thaliana

La presente tesis doctoral se enmarca dentro del tema de la homeostasis de cationes en células vegetales. El conocimiento de los transportadores involucrados en la homeostasis de cationes y sus mecanismos de regulación, puede tener aplicaciones biotecnológicas para el desarrollo de plantas tolerantes a estreses abióticos como la salinidad o el pH ácido. El objetivo general del presente trabajo es determinar y estudiar los mecanismos implicados en la homeostasis de pH en Arabidopsis thaliana. La metodología empleada ha consistido, por un lado, en la realización de micromatrices para estudiar la respuesta transcripcional al estrés por ácido acético. Por otro lado, se ha llevado a cabo un rastreo genético para encontrar genes determinantes de la tolerancia a este estrés. Los resultados del análisis transcripcional han servido para identificar posibles genes cuya expresión es relevante para la homeostasis de pH. De hecho, estos resultados han servido de base para otra tesis doctoral realizada en el departamento. Los resultados del rastreo genético han permitido aislar y caracterizar un mutante dominante negativo (wat1-1D: "weak acid tolerant") que expresa una versión truncada de la adaptina ß3 de A. thaliana y que posee una alteración en la homeostasis de pH y de otros cationes monovalentes, como el sodio o el potasio.Niñoles Rodenes, R. (2011). Biología molecular de la regulación de la homeostasis de pH en Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11301Palanci