NIN-like protein7 and PROTEOLYSIS6 functional interaction enhances tolerance to sucrose, ABA, and submergence.

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.

Post-Translational Modifications of Nitrate Reductases Autoregulates Nitric Oxide Biosynthesis in Arabidopsis.

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

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

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.

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

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.

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

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.

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

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.

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

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.

Nitric Oxide Controls Constitutive Freezing Tolerance in Arabidopsis by Attenuating the Levels of Osmoprotectants, Stress-Related Hormones and Anthocyanins.

Plant tolerance to freezing temperatures is governed by endogenous constitutive components and environmental inducing factors. Nitric oxide (NO) is one of the endogenous components that participate in freezing tolerance regulation. A combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants suggests that NO acts attenuating the production and accumulation of osmoprotective and regulatory metabolites, such as sugars and polyamines, stress-related hormones, such as ABA and jasmonates, and antioxidants, such as anthocyanins and flavonoids. Accordingly, NO-deficient plants are constitutively more freezing tolerant than wild type plants.