Arabidopsis VQ motif-containing proteins VQ1 and VQ10 interact with plastidial 1-deoxy-D-xylulose-5-phosphate synthase.

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein-protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively.

Enhanced meristem development, tolerance to oxidative stress and hyposensitivity to nitric oxide in the hypermorphic vq10-H mutant in AtVQ10 gene.

Nitric oxide (NO) and reactive oxygen are common factors in multiple plant responses to stress, and their involvement in hypoxia-triggered responses is key to ensure growth under adverse environmental conditions. Here, we analyse the regulatory functions exerted by hypoxia-, NO- and oxidative stress-inducible Arabidopsis gene coding for the VQ motif-containing protein 10 (VQ10). A hypermorphic vq10-H mutant allowed identifying VQ10-exerted regulation on root and shoot development as well as its role in regulating responses to NO and oxidative stress. Enhanced VQ10 expression in vq10-H plants led to enhanced elongation of the primary root, and increased root cell division and meristem size during early postgermination development. In shoots, VQ10 activation of cell division was counteracted by WRKY33-exerted repression, thus leading to a dwarf bushy phenotype in plants with enhanced VQ10 expression in a wrky33 knock-out background. Low number of differentially expressed genes were identified when vq10-H versus Col-0 plants were compared either under normoxia or hypoxia. vq10-H and VQ10ox plants displayed less tolerance to submergence but, in turn, were more tolerant to oxidative stress and less sensitive to NO than wild-type plants. VQ10 could be a node integrating redox-related regulation on development and stress responses.

Thioredoxin TRXo1 is involved in ABA perception via PYR1 redox regulation.

Abscisic acid (ABA) plays a fundamental role in plant growth and development processes such as seed germination, stomatal response or adaptation to stress, amongst others. Increases in the endogenous ABA content is recognized by specific receptors of the PYR/PYL/RCAR family that are coupled to a phosphorylation cascade targeting transcription factors and ion channels. Just like other receptors of the family, nuclear receptor PYR1 binds ABA and inhibits the activity of type 2C phosphatases (PP2Cs), thus avoiding the phosphatase-exerted inhibition on SnRK2 kinases, positive regulators which phosphorylate targets and trigger ABA signalling. Thioredoxins (TRXs) are key components of cellular redox homeostasis that regulate specific target proteins through a thiol-disulfide exchange, playing an essential role in redox homeostasis, cell survival, and growth. In higher plants, TRXs have been found in almost all cellular compartments, although its presence and role in nucleus has been less studied. In this work, affinity chromatography, Dot-blot, co-immunoprecipitation, and bimolecular fluorescence complementation assays allowed us to identify PYR1 as a new TRXo1 target in the nucleus. Studies on recombinant HisAtPYR1 oxidation-reduction with wild type and site-specific mutagenized forms showed that the receptor underwent redox regulation involving changes in the oligomeric state in which Cys30 and Cys65 residues were implied. TRXo1 was able to reduce previously-oxidized inactive PYR1, thus recovering its capacity to inhibit HAB1 phosphatase. In vivo PYR1 oligomerization was dependent on the redox state, and a differential pattern was detected in KO and over-expressing Attrxo1 mutant plants grown in the presence of ABA compared to WT plants. Thus, our findings suggest the existence of a redox regulation of TRXo1 on PYR1 that may be relevant for ABA signalling and had not been described so far.

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.

Valine-Glutamine Proteins in Plant Responses to Oxygen and Nitric Oxide.

Multigene families coding for valine-glutamine (VQ) proteins have been identified in all kind of plants but chlorophytes. VQ proteins are transcriptional regulators, which often interact with WRKY transcription factors to regulate gene expression sometimes modulated by reversible phosphorylation. Different VQ-WRKY complexes regulate defense against varied pathogens as well as responses to osmotic stress and extreme temperatures. However, despite these well-known functions, new regulatory activities for VQ proteins are still to be explored. Searching public Arabidopsis thaliana transcriptome data for new potential targets of VQ-WRKY regulation allowed us identifying several VQ protein and WRKY factor encoding genes that were differentially expressed in oxygen-related processes such as responses to hypoxia or ozone-triggered oxidative stress. Moreover, some of those were also differentially regulated upon nitric oxide (NO) treatment. These subsets of VQ and WRKY proteins might combine into different VQ-WRKY complexes, thus representing a potential regulatory core of NO-modulated and O2-modulated responses. Given the increasing relevance that gasotransmitters are gaining as plant physiology regulators, and particularly considering the key roles exerted by O2 and NO in regulating the N-degron pathway-controlled stability of transcription factors, VQ and WRKY proteins could be instrumental in regulating manifold processes 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.

Nitric oxide triggers a transient metabolic reprogramming in Arabidopsis.

Nitric oxide (NO) regulates plant growth and development as well as responses to stress that enhanced its endogenous production. Arabidopsis plants exposed to a pulse of exogenous NO gas were used for untargeted global metabolomic analyses thus allowing the identification of metabolic processes affected by NO. At early time points after treatment, NO scavenged superoxide anion and induced the nitration and the S-nitrosylation of proteins. These events preceded an extensive though transient metabolic reprogramming at 6 h after NO treatment, which included enhanced levels of polyamines, lipid catabolism and accumulation of phospholipids, chlorophyll breakdown, protein and nucleic acid turnover and increased content of sugars. Accordingly, lipid-related structures such as root cell membranes and leaf cuticle altered their permeability upon NO treatment. Besides, NO-treated plants displayed degradation of starch granules, which is consistent with the increased sugar content observed in the metabolomic survey. The metabolic profile was restored to baseline levels at 24 h post-treatment, thus pointing up the plasticity of plant metabolism in response to nitroxidative stress conditions.

FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway.

Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants.

Abscisic acid (ABA) is a phytohormone that inhibits growth and enhances adaptation to stress in plants. ABA perception and signaling rely on its binding to receptors of the pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) family, the subsequent inhibition of clade A type 2C protein phosphatases (PP2Cs), and the phosphorylation of ion channels and transcription factors by protein kinases of the SnRK2 family. Nitric oxide (NO) may inhibit ABA signaling because NO-deficient plants are hypersensitive to ABA. Regulation by NO often involves posttranslational modification of proteins. Mass spectrometry analysis of ABA receptors expressed in plants and recombinant receptors modified in vitro revealed that the receptors were nitrated at tyrosine residues and S-nitrosylated at cysteine residues. In an in vitro ABA-induced, PP2C inhibition assay, tyrosine nitration reduced receptor activity, whereas S-nitrosylated receptors were fully capable of ABA-induced inhibition of the phosphatase. PYR/PYL/RCAR proteins with nitrated tyrosine, which is an irreversible covalent modification, were polyubiquitylated and underwent proteasome-mediated degradation. We propose that tyrosine nitration, which requires NO and superoxide anions, is a rapid mechanism by which NO limits ABA signaling under conditions in which NO and reactive oxygen species are both produced.

Identification of transcription factors potentially involved in the juvenile to adult phase transition in Citrus.

BACKGROUND AND AIMS: The juvenile to adult transition (JAT) in higher plants is required for them to reach reproductive competence. However, it is a poorly understood process in woody plants, where only a few genes have been definitely identified as being involved in this transition. This work aims at increasing our understanding of the mechanisms regulating the JAT in citrus. METHODS: Juvenile and adult plants from Pineapple sweet orange (Citrus sinensis) and Rough lemon (C. jambhiri) were used to screen for differentially expressed transcription factors (TFs) using a 1·15K microarray developed on the basis of the CitrusTF database. Murcott tangor (C. reticulata × C. sinensis) and Duncan grapefruit (C. paradisi) were incorporated into the quantitative real-time reverse transcription-PCR validation in order to select those genes whose phase-specific regulation was common to the four species. KEY RESULTS: A browsable web database has been created with information about the structural and functional annotation related to 1152 unigenes of putative citrus TFs (CTFs). This database constitutes a valuable resource for research on transcriptional regulation and comparative genomics. Moreover, a microarray has been developed and used that contains these putative CTFs, in order to identify eight genes that showed differential expression in juvenile and adult meristems of four different species of citrus. Those genes have been characterized, and their expression pattern in vegetative and reproductive tissues has been analysed. Four of them are MADS-box genes, a family of TFs involved in developmental processes, whereas another one resembles MADS-box genes but lacks the MADS box itself. The other three showed high partial sequence similarity restricted to specific Arabidopsis protein domains but negligible outside those domains. CONCLUSIONS: The work presented here indicates that the JAT in citrus could be controlled by mechanisms that are in part common to those of Arabidopsis, but also somehow different, since specific factors without Arabidopsis orthologues have also been characterized. The potential involvement of the genes in the JAT is discussed.