Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana

El óxido nítrico (NO) es una molécula gaseosa altamente reactiva que regula el crecimiento y el desarrollo de las plantas así como sus respuestas de defensa. El NO se produce principalmente a partir de nitrito por las nitrato reductasas (NRs) en balance con las nitrito reductasas (NiRs), y es percibido a través de un mecanismo en el que está involucrada la proteólisis dirigida por la secuencia aminoterminal del grupo VII de los factores de transcripción ERF (ERFVIIs). El NO ejerce especialmente su función señalizadora al causar modificaciones postraduccionales en las proteínas y alterar su función, estructura y/o estabilidad. Por estos medios y en colaboración con distintas rutas de señalización fitohormonales, el NO es capaz de regular un amplio abanico de procesos celulares en plantas, incluyendo aquellos relacionados con la adquisición de tolerancia a la congelación. Utilizando Arabidopsis thaliana como planta modelo, en este trabajo se descubrió que el NO puede regular su propia biosíntesis, puesto que las enzimas NRs y NiRs fueron reguladas por tres factores principales: señalización inducida por nitrato y controlada por la función del factor de transcripción NIN-like protein 7 (NLP7), la proteólisis dirigida por la secuencia aminoterminal, y la degradación mediada por el proteasoma, probablemente ocasionada por modificaciones postraduccionales relacionadas con el NO. Adicionalmente, se descubrió que el factor de transcripción ERFVII RAP2.3 regula negativamente tanto la biosíntesis de NO como las respuestas que desencadena a través de un mecanismo similar a un reóstato en el que están involucradas ramas específicas relacionadas con el NO de las rutas de señalización de jasmonato y ácido abscísico. Por otro lado, una caracterización metabolómica y transcriptómica combinada de plantas mutantes nia1,2noa1-2 deficientes en NO y plantas fumigadas con NO permitió desentrañar una serie de mecanismos que están controlados por NO. En primer lugar, la percepción de NO en los hipocotilos requeriría varias hormonas para ser completada, como fue confirmado por los rastreos de acortamiento de hipocotilo por NO con mutantes relacionados con hormonas y la colección TRANSPLANTA de líneas transgénicas que expresan condicionalmente factores de transcripción de Arabidopsis. En segundo lugar, dosis elevadas de NO causan una reprogramación masiva aunque transitoria de los metabolismos primario y secundario, incluyendo la alteración del estado redox celular, la alteración de la permeabilidad de estructuras lipídicas y el recambio de proteínas y ácidos nucleicos. Por último, se descubrió que el NO previene el desarrollo de la tolerancia a congelación bajo condiciones no estresantes de temperatura, mientras que resulta esencial para la aclimatación a frío desencadenada por bajas temperaturas que conduce a una tolerancia mejorada a congelación. El NO conseguiría esta modulación afinada de la activación de respuestas relacionadas con frío al coordinar la acumulación de diferentes metabolitos y hormonas. En conjunto, este trabajo arroja luz sobre los mecanismos mediante los cuales, al interactuar con varias rutas señalizadoras y metabólicas, el NO puede regular distintos procesos clave de la fisiología vegetal.L'òxid nítric (NO) és una molècula gasosa altament reactiva que regula el creixement i desenvolupament de les plantes així com les seves respostes de defensa. El NO es produeix principalment a partir de nitrit per les nitrat reductases (NRs) en balanç amb les nitrit reductases (NiRs), i és percebut a traves d'un mecanisme que inclou la proteòlisi dirigida per la seqüència aminoterminal del grup VII dels factors de transcripció ERF (ERFVII). El NO exerceix la seva funció senyalitzadora majoritàriament al provocar modificacions postraduccionals en les proteïnes i alterar la seva funció, estructura i/o estabilitat. Mitjançant aquestes modificacions i en col·laboració amb distintes rutes de senyalització fitohormonals, el NO es capaç de regular un ampli espectre de processos cel·lulars en plantes, inclosos aquells relacionats amb l'adquisició de tolerància a la congelació. Emprant Arabidopsis thaliana com a planta model, en aquest treball es va descobrir que el NO regula la seva pròpia biosíntesi, donat que els enzims NRs i NiRs foren regulades per tres factors principals: senyalització induïda per nitrat i controlada per la funció del factor de transcripció NIN-like protein 7 (NLP7), la proteòlisi dirigida per la seqüència aminoterminal, i la degradació mitjançant el proteasoma, probablement a causa de modificacions postraduccionals relacionades amb el NO. A més, es va descobrir que el factor de transcripció ERFVII RAP2.3 regula negativament tant la biosíntesi de NO com les respostes que desencadena aquest a través d'un mecanisme similar a un reòstat en el que estan involucrades branques específiques de les rutes de senyalització de jasmonat i àcid abscísic relacionades amb el NO. Per altre costat, una caracterització metabolòmica i transcriptòmica combinada de plantes mutants nia1,2noa1-2 deficients en NO i plantes fumigades amb NO va permetre desentranyar una sèrie de mecanismes que estan controlats per NO. En primer lloc, la percepció de NO en els hipocòtils requeriria de varies hormones, com fou confirmat pels rastrejos d'acurtament d'hipocòtil per NO amb mutants relacionats amb hormones i la col·lecció TRANSPLANTA de línies transgèniques d'expressió condicional de factors de transcripció d'Arabidopsis. En segon lloc, dosis elevades de NO causen una reprogramació massiva, encara que transitòria, dels metabolismes primari i secundari, incloent l'alteració de l'estat redox cel·lular, canvis en la permeabilitat de estructures lipídiques i el recanvi de proteïnes i àcids nucleics. Per últim, es va descobrir que el NO prevé el desenvolupament de la tolerància a congelació en condicions no estressants de temperatura, mentre que resulta essencial per a l'aclimatació a fred induïda per baixes temperatures que condueix a una tolerància millorada a congelació. El NO aconseguiria aquesta modulació minuciosa de l'activació de les respostes relacionades amb fred al coordinar l'acumulació de diferents metabòlits i hormones. En conjunt, aquest treball clarifica els mecanismes pels quals el NO pot regular distints processos clau de la fisiologia vegetal al interactuar amb varies rutes senyalitzadores i metabòliques.Nitric oxide (NO) is a highly reactive gaseous molecule that regulates plant growth and development as well as defense responses. NO is mainly produced from nitrite by nitrate reductases (NRs) in balance with nitrite reductases (NiRs), and is sensed through a mechanism involving the N-end rule pathway-mediated proteolysis of the group VII of ERF transcription factors (ERFVIIs). NO especially exerts its signaling function by triggering post-translational modifications in proteins and altering their function, structure and/or stability. By these means and in collaboration with different phytohormone signaling pathways, NO is capable of regulating a wide array of cell processes in plants, including those related to the acquirement of freezing tolerance. By using Arabidopsis thaliana as model plant, during the development of this work it was found that NO can regulate its own biosynthesis, as NRs and NiR enzymes were regulated by three main factors: nitrate-induced signaling controlled by the function of the NIN-like protein 7 (NLP7) transcription factor, N-end rule proteolytic pathway, and proteasome-mediated degradation, likely triggered by NO-related post-translational modifications. In addition, the ERFVII transcription factor RAP2.3 was found to negatively regulate both the NO biosynthesis and their triggered responses through a rheostat-like mechanism that involves specific NO-related branches of jasmonate and abscisic acid signaling pathways. On the other hand, a combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants and NO-fumigated plants allowed to unravel a number of mechanisms that are controlled by NO. First, NO perception in hypocotyls would require various hormones to be fulfilled as it was confirmed by NO-triggered hypocotyl shortening screenings with hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis transcription factors. Second, high NO doses caused a massive but transient reprogramming of primary and secondary metabolism, including alteration of the cellular redox status, alteration of the permeability of lipidic structures or turnover of proteins and nucleic acids. Lastly, NO was found to prevent the development of freezing tolerance under non-stress temperature conditions, while being essential for the low temperature stress-triggered cold acclimation that leads to enhanced freezing tolerance. NO would achieve this fine-tuned modulation of the activation of the cold-related responses by coordinating the accumulation of different metabolites and hormones. Altogether, this work sheds light on the mechanisms by which, by interacting with various signaling and metabolic pathways, NO can regulate several key processes of plant physiology.Costa Broseta, Á. (2018). Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825TESI

Present knowledge and controversies, deficiencies and misconceptions on nitric oxide synthesis, sensing and signaling in plants

[EN] After 30 years of intensive work, nitric oxide (NO) has just started to be characterized as a relevant regulatory molecule on plant development and responses to stress. Its reactivity as a free radical determines its mode of action as an inducer of posttranslational modifications of key target proteins through cysteine S-nitrosylation and tyrosine nitration. Many of the NO-triggered regulatory actions are exerted in tight coordination with phytohormone signaling. This review not only summarizes and updates the information accumulated on how NO is synthesized, sensed, and transduced in plants but also makes emphasis on controversies, deficiencies, and misconceptions that are hampering our present knowledge on the biology of NO in plants. The development of noninvasive accurate tools for the endogenous NO quantitation as well as the implementation of genetic approaches that overcome misleading pharmacological experiments will be critical for getting significant advances in better knowledge of NO homeostasis and regulatory actions in plants.Ministerio de Ciencia e Innovacion, Grant/Award Numbers: BIO2014-56067-P and BIO2017-82945-P; Government of Spain MINECO, Grant/Award Numbers: BIO2017-82945-P and BIO2014-56067-PLeon Ramos, J.; Costa-Broseta, Á. (2020). Present knowledge and controversies, deficiencies and misconceptions on nitric oxide synthesis, sensing and signaling in plants. Plant Cell & Environment. 43(1):1-15. https://doi.org/10.1111/pce.13617S11543

RAP2.3 negatively regulates nitric oxide biosynthesis and related responses through a rheostat-like mechanism in Arabidopsis

[EN] Nitric oxide (NO) is sensed through a mechanism involving the degradation of group-VII ERF transcription factors (ERFVIIs) that is mediated by the N-degron pathway. However, the mechanisms regulating NO homeostasis and downstream responses remain mostly unknown. To explore the role of ERFVIIs in regulating NO production and signaling, genome-wide transcriptome analyses were performed on single and multiple erfvii mutants of Arabidopsis following exposure to NO. Transgenic plants overexpressing degradable or non-degradable versions of RAP2.3, one of the five ERFVIIs, were also examined. Enhanced RAP2.3 expression attenuated the changes in the transcriptome upon exposure to NO, and thereby acted as a brake for NO-triggered responses that included the activation of jasmonate and ABA signaling. The expression of non-degradable RAP2.3 attenuated NO biosynthesis in shoots but not in roots, and released the NO-triggered inhibition of hypocotyl and root elongation. In the guard cells of stomata, the control of NO accumulation depended on PRT6-triggered degradation of RAP2.3 more than on RAP2.3 levels. RAP2.3 therefore seemed to work as a molecular rheostat controlling NO homeostasis and signaling. Its function as a brake for NO signaling was released upon NO-triggered PRT6-mediated degradation, thus allowing the inhibition of growth, and the potentiation of jasmonate- and ABA-related signaling.We would like to acknowledge Lorena Latorre (Genomic Service at IBMCP) for her support in the hybridizing Agilent microarrays. This work was supported by grants BIO2014-56067-P and BIO2017-82945-P from the Spanish Ministry of Economy, Industry and Competitiveness, and by FEDER funds. The authors declare no conflicts of interest.Leon Ramos, J.; Costa-Broseta, Á.; Castillo López Del Toro, MC. (2020). RAP2.3 negatively regulates nitric oxide biosynthesis and related responses through a rheostat-like mechanism in Arabidopsis. 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Combinatorial interaction network of abscisic acid receptors and coreceptors fromArabidopsis thaliana. Proceedings of the National Academy of Sciences, 114(38), 10280-10285. doi:10.1073/pnas.1706593114Tsai, Y.-C., Delk, N. A., Chowdhury, N. I., & Braam, J. (2007). Arabidopsis Potential Calcium Sensors Regulate Nitric Oxide Levels and the Transition to Flowering. Plant Signaling & Behavior, 2(6), 446-454. doi:10.4161/psb.2.6.4695Tsutsui, T., Kato, W., Asada, Y., Sako, K., Sato, T., Sonoda, Y., … Yamaguchi, J. (2009). DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis. Journal of Plant Research, 122(6), 633-643. doi:10.1007/s10265-009-0252-6Van Verk, M. C., Bol, J. F., & Linthorst, H. J. (2011). WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes. BMC Plant Biology, 11(1). doi:10.1186/1471-2229-11-89Varshavsky, A. (2019). N-degron and C-degron pathways of protein degradation. 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The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors. Journal of Biological Chemistry, 293(30), 11786-11795. doi:10.1074/jbc.ra118.003496Williams, B. P., Pignatta, D., Henikoff, S., & Gehring, M. (2015). Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat. PLOS Genetics, 11(3), e1005142. doi:10.1371/journal.pgen.1005142Zhang, Y., Wang, L., Liu, Y., Zhang, Q., Wei, Q., & Zhang, W. (2006). Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta, 224(3), 545-555. doi:10.1007/s00425-006-0242-zZottini, M., Costa, A., De Michele, R., Ruzzene, M., Carimi, F., & Lo Schiavo, F. (2007). Salicylic acid activates nitric oxide synthesis in Arabidopsis. Journal of Experimental Botany, 58(6), 1397-1405. doi:10.1093/jxb/erm00

Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis

[EN] Plant growth is the result of the coordinated photosynthesis-mediated assimilation of oxidized forms of C, N and S. Nitrate is the predominant N source in soils and its reductive assimilation requires the successive activities of soluble cytosolic NADH-nitrate reductases (NR) and plastid stroma ferredoxin-nitrite reductases (NiR) allowing the conversion of nitrate to nitrite and then to ammonium. However, nitrite, instead of being reduced to ammonium in plastids, can be reduced to nitric oxide (NO) in mitochondria, through a process that is relevant under hypoxic conditions, or in the cytoplasm, through a side-reaction catalyzed by NRs. We use a loss-of-function approach, based on CRISPR/Cas9-mediated genetic edition, and gain-of-function, using transgenic overexpressing HA-tagged Arabidopsis NiR1 to characterize the role of this enzyme in controlling plant growth, and to propose that the NO-related post-translational modifications, by S-nitrosylation of key C residues, might inactivate NiR1 under stress conditions. NiR1 seems to be a key target in regulating nitrogen assimilation and NO homeostasis, being relevant to the control of both plant growth and performance under stress conditions. Because most higher plants including crops have a single NiR, the modulation of its function might represent a relevant target for agrobiotechnological purposes.This research was funded by BIO2014-56067-P and BIO2017-82945-P grants from the Spanish Ministry of Economy, Industry and Competitiveness and Fondo Europeo de Desarrollo Regional (FEDER) funds.Costa-Broseta, Á.; Castillo López Del Toro, MC.; Leon Ramos, J. (2020). Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis. International Journal of Molecular Sciences. 21(19):1-13. https://doi.org/10.3390/ijms211972701132119Solomonson, L. P., & Barber, M. J. (1990). Assimilatory Nitrate Reductase: Functional Properties and Regulation. Annual Review of Plant Physiology and Plant Molecular Biology, 41(1), 225-253. doi:10.1146/annurev.pp.41.060190.001301Knaff, D. B., & Hirasawa, M. (1991). Ferredoxin-dependent chloroplast enzymes. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1056(2), 93-125. doi:10.1016/s0005-2728(05)80277-4Wang, R., Xing, X., & Crawford, N. (2007). Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots. Plant Physiology, 145(4), 1735-1745. doi:10.1104/pp.107.108944Tanaka, S., Ida, S., Irifune, K., Oeda, K., & Morikawa, H. (1994). Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana. DNA Sequence, 5(1), 57-61. doi:10.3109/10425179409039705LEA, P. J., & MIFLIN, B. J. (1974). Alternative route for nitrogen assimilation in higher plants. Nature, 251(5476), 614-616. doi:10.1038/251614a0Gupta, K. J., & Igamberdiev, A. U. (2011). The anoxic plant mitochondrion as a nitrite: NO reductase. Mitochondrion, 11(4), 537-543. doi:10.1016/j.mito.2011.03.005Rockel, P., Strube, F., Rockel, A., Wildt, J., & Kaiser, W. M. (2002). Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. Journal of Experimental Botany, 53(366), 103-110. doi:10.1093/jexbot/53.366.103Bender, D., & Schwarz, G. (2018). Nitrite-dependent nitric oxide synthesis by molybdenum enzymes. FEBS Letters, 592(12), 2126-2139. doi:10.1002/1873-3468.13089Kolbert, Z., Barroso, J. B., Brouquisse, R., Corpas, F. J., Gupta, K. J., Lindermayr, C., … Hancock, J. T. (2019). A forty year journey: The generation and roles of NO in plants. Nitric Oxide, 93, 53-70. doi:10.1016/j.niox.2019.09.006Astier, J., & Lindermayr, C. (2012). Nitric Oxide-Dependent Posttranslational Modification in Plants: An Update. International Journal of Molecular Sciences, 13(12), 15193-15208. doi:10.3390/ijms131115193Jain, P., & Bhatla, S. C. (2018). Molecular mechanisms accompanying nitric oxide signalling through tyrosine nitration and S-nitrosylation of proteins in plants. Functional Plant Biology, 45(2), 70. doi:10.1071/fp16279Calatrava, V., Chamizo-Ampudia, A., Sanz-Luque, E., Ocaña-Calahorro, F., Llamas, A., Fernandez, E., & Galvan, A. (2017). How Chlamydomonas handles nitrate and the nitric oxide cycle. Journal of Experimental Botany, 68(10), 2593-2602. doi:10.1093/jxb/erw507De Montaigu, A., Sanz-Luque, E., Galván, A., & Fernández, E. (2010). A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas  . The Plant Cell, 22(5), 1532-1548. doi:10.1105/tpc.108.062380Kim, J. Y., Kwon, Y. J., Kim, S.-I., Kim, D. Y., Song, J. T., & Seo, H. S. (2016). Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.01161Castillo, M.-C., Coego, A., Costa-Broseta, Á., & León, J. (2018). Nitric oxide responses in Arabidopsis hypocotyls are mediated by diverse phytohormone pathways. Journal of Experimental Botany, 69(21), 5265-5278. doi:10.1093/jxb/ery286Wang, J., Wang, Y., Lv, Q., Wang, L., Du, J., Bao, F., & He, Y.-K. (2017). Nitric oxide modifies root growth by S-nitrosylation of plastidial glyceraldehyde-3-phosphate dehydrogenase. Biochemical and Biophysical Research Communications, 488(1), 88-94. doi:10.1016/j.bbrc.2017.05.012Chen, Z. J., & Sun, L. J. (2009). Nonproteolytic Functions of Ubiquitin in Cell Signaling. Molecular Cell, 33(3), 275-286. doi:10.1016/j.molcel.2009.01.014Thrower, J. S. (2000). Recognition of the polyubiquitin proteolytic signal. The EMBO Journal, 19(1), 94-102. doi:10.1093/emboj/19.1.94Chu, C.-C., & Li, H. (2018). Developmental regulation of protein import into plastids. Photosynthesis Research, 138(3), 327-334. doi:10.1007/s11120-018-0546-4Hirasawa, M., Tollin, G., Salamon, Z., & Knaff, D. B. (1994). Transient kinetic and oxidation-reduction studies of spinach ferrodoxin: nitrate oxidoreductase. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1185(3), 336-345. doi:10.1016/0005-2728(94)90249-6Hirasawa, M., Tripathy, J. N., Somasundaram, R., Johnson, M. K., Bhalla, M., Allen, J. P., & Knaff, D. B. (2009). The Interaction of Spinach Nitrite Reductase with Ferredoxin: A Site-Directed Mutation Study. Molecular Plant, 2(3), 407-415. doi:10.1093/mp/ssn098Y., M.-G.-T., P., R., T., M., I., Q., M., L., W., K., & J., M.-G. (2002). Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco. Planta, 215(5), 708-715. doi:10.1007/s00425-002-0816-3Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xChiu, J., Tillett, D., Dawes, I. W., & March, P. E. (2008). Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb. Journal of Microbiological Methods, 73(2), 195-198. doi:10.1016/j.mimet.2008.02.013Wang, Z.-P., Xing, H.-L., Dong, L., Zhang, H.-Y., Han, C.-Y., Wang, X.-C., & Chen, Q.-J. (2015). Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biology, 16(1). doi:10.1186/s13059-015-0715-0Davenport, S., Le Lay, P., & Sanchez-Tamburrrino, J. P. (2015). Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase. 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NIN-like protein7 and PROTEOLYSIS6 functional interaction enhances tolerance to sucrose, ABA, and submergence

[EN] Nitrate (NO3) assimilation and signaling regulate plant growth through the relevant function of the transcription factor NIN-like Protein7 (NLP7). NO3 is also the main source for plants to produce nitric oxide (NO), which regulates growth and stress responses. NO-mediated regulation requires efficient sensing via the PROTEOLYSIS6 (PRT6)-mediated proteasome-triggered degradation of group VII of ethylene response transcription factors through the Cys/Arg N-degron pathway. The convergence of NO3 signaling and N-degron proteolysis on NO-mediated regulation remains largely unknown. Here, we investigated the functional interaction between NLP7 and PRT6 using Arabidopsis (Arabidopsis thaliana) double prt6 nlp7 mutant plants as well as complementation lines overexpressing NLP7 in different mutant genetic backgrounds. prt6 nlp7 mutant plants displayed several potentiated prt6 characteristic phenotypes, including slower vegetative growth, increased NO content, and diminished tolerance to abiotic stresses such as high-sucrose concentration, abscisic acid, and hypoxia-reoxygenation. Although NLP7 has an N-terminus that could be targeted by the N-degron proteolytic pathway, it was not a PRT6 substrate. The potential PRT6- and NO-regulated nucleocytoplasmic translocation of NLP7, which is likely modulated by posttranslational modifications, is proposed to act as a regulatory loop to control NO homeostasis and action.This work was supported by MINECO from Spain grant (BIO2017-82945-P), CSIC (2020AEP055), Generalitat Valenciana (PROMETEO/2019/021 grant), and FEDER funds from European Union.Castillo López Del Toro, MC.; Costa-Broseta, Á.; Gayubas, B.; Leon Ramos, J. (2021). NIN-like protein7 and PROTEOLYSIS6 functional interaction enhances tolerance to sucrose, ABA, and submergence. Plant Physiology. 187(4):2731-2748. https://doi.org/10.1093/plphys/kiab38227312748187

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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Nitric oxide responses in Arabidopsis hypocotyls are mediated by diverse phytohormone pathways

[EN] Plants are often exposed to high levels of nitric oxide (NO) that affects development and stress-triggered responses. However, the way in which plants sense NO is still largely unknown. Here we combine the analysis of early changes in the transcriptome of plants exposed to a short acute pulse of exogenous NO with the identification of transcription factors (TFs) involved in NO sensing. The NO-responsive transcriptome was enriched in hormone homeostasis and signaling-related genes. To assess events involved in NO sensing in hypocotyls, we used a functional sensing assay based on the NO-induced inhibition of hypocotyl elongation in etiolated seedlings. Hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis TFs were screened for NO-triggered hypocotyl shortening. These approaches allowed the identification of hormone-related TFs, ethylene perception and signaling, strigolactone biosynthesis and signaling, and salicylate production and accumulation that are essential for or modulate hypocotyl NO sensing. Moreover, NO inhibits hypocotyl elongation through the positive and negative regulation of some abscisic acid (ABA) receptors and transcripts encoding brassinosteroid signaling components thereby also implicating these hormones in NO sensing.This work was supported by grants BIO2014-56067-P and BIO2017-82945-P from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER funds. We thank the Genomics Unit of the Centro Nacional de Biotecnologia (CNB-CSIC, Madrid, Spain) for microarray processing. We gratefully acknowledged the kind donation of mutant seeds: Roberto Solano and Andrea Chini (CNB, Madrid, Spain) for the jaz and myc mutants; Paul Verslues (Institute of Plant and Microbial Biology, Academia Sinica, Taiwan) for the hai mutants; Javier Agusti, Pablo Tornero, and Pedro Rodriguez (IBMCP, Valencia, Spain) for the max, sid2eds5nahG, and pyr/pyl mutants, respectively; and Hiroaki Fujii (University of Turku, Finland) for the snrk2.3 and 2.9 mutants.Castillo López Del Toro, MC.; Coego Gonzalez, A.; Costa-Broseta, Á.; Leon Ramos, J. (2018). Nitric oxide responses in Arabidopsis hypocotyls are mediated by diverse phytohormone pathways. Journal of Experimental Botany. 69(21):5265-5278. https://doi.org/10.1093/jxb/ery286S52655278692

Identificación y caracterización funcional del transcriptoma regulado por óxido nítrico en A. thaliana

[EN] Nitric oxide (NO) regulates developmental and stress-related plant processes through still largely undefined mechanisms. To study the effect of deficient NO biosynthesis on genome-wide Arabidopsis thaliana transcriptome, the nia1nia2noa1-2 mutant plants, impaired in the nitrate reductase (NR/NIA)- and nitric oxide associated 1 (NOA1)-related NO biosynthesis, were compared to wild type plants by using microarray analyses. Gene Ontology (GO) analyses on differentially expressed genes pointed to altered biosynthesis and metabolism of phenylpropanoids and amino acid derivatives. Besides, nia1nia2noa1-2 roots were hypersensitive to ABA, ethylene and cytokinins and hyposensitive to auxins. Further microarray-based analyses of parental nia1nia2 and noa1-2 mutants versus Col-0 plants allowed defining a core of 66 genes, 46 down-regulated and 20 up-regulated in all tested NO-deficient plants. The in silico analyses of their gene expression patterns and the lack of evidence in phenotypic assays of a transcription factor among these genes being a master regulator of NO-regulated responses in Arabidopsis suggest an extensive co-regulation between NO and ethylene and anti-regulation with ABA, auxins, salicylates and cytokinins, pointing to a key role of NO in hormone signaling.[ES] El óxido nítrico (NO) regula procesos de desarrollo y relacionados con el estrés en plantas a través de mecanismos en gran medida aún por definir. Para estudiar el efecto de la biosíntesis deficiente de NO en el transcriptoma completo de Arabidopsis thaliana, las plantas mutantes nia1nia2noa1-2, con la biosíntesis de NO a través de la nitrato reductasa (NR/NIA) y de la proteína asociada a óxido nítrico (NOA1) bloqueadas, se compararon con plantas silvestres mediante análisis de micromatrices. Los análisis de ontologías génicas (GO) en los genes diferencialmente expresados desvelaron alteraciones en la biosíntesis y el metabolismo de derivados de aminoácidos y fenilpropanoides. Además, las raíces de nia1nia2no1-2 resultaron ser hipersensibles a ABA, etileno y citoquininas e hiposensibles a auxinas. Análisis adicionales de micromatrices de los parentales mutantes nia1nia2 y noa1-2 contra plantas Col-0 permitieron definir un núcleo de 66 genes, 46 regulados a la baja y 20 a la alza, en todas las plantas testadas deficientes en NO. Los análisis in silico de sus patrones de expresión génica y la falta de evidencia en ensayos fenotípicos de que alguno de los factores e transcripción entre estos genes se comporte como un regulador maestro de la respuesta a NO en Arabidopsis sugieren una extensa corregulación entre el NO y el etileno y una anti-regulación con ABA, auxinas, salicilatos y citoquininas, apuntando a un papel destacada del NO en la señalización hormonal.Costa Broseta, Á. (2015). Identificación y caracterización funcional del transcriptoma regulado por óxido nítrico en A. thaliana. http://hdl.handle.net/10251/6422