Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors.

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

Distinct branches of the N-end rule pathway modulate the plant immune response.

The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

Sumoylation and phosphorylation: hidden and overt links.

Post-translational modifications are essential mediators between stimuli from development or the environment and adaptive transcriptional patterns. Recent data allow a first glimpse at how two modifications, phosphorylation and sumoylation, act interdependently to modulate stress responses. In particular, many components of the SUMO conjugation system are phosphoproteins, and some regulators and enzymes of protein phosphorylation can be sumoylated. Equally important, however, a number of proteins can be subject to both modifications. These substrates also have the capacity to connect stimuli transmitted via sumoylation with those transmitted via phosphorylation. As a prime example, we review data suggesting that nitrate reductase is a hub that integrates cues from these two modifications. Powerful proteomics approaches allowed the identification of additional common substrates, paving the way for studies to understand, on a broader basis, the cross-talk of phosphorylation with sumoylation and how it contributes to plant growth.

Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6.

Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore, we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen-sensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

Group VII Ethylene Response Factors Coordinate Oxygen and Nitric Oxide Signal Transduction and Stress Responses in Plants.

The group VII ethylene response factors (ERFVIIs) are plant-specific transcription factors that have emerged as important regulators of abiotic and biotic stress responses, in particular, low-oxygen stress. A defining feature of ERFVIIs is their conserved N-terminal domain, which renders them oxygen- and nitric oxide (NO)-dependent substrates of the N-end rule pathway of targeted proteolysis. In the presence of these gases, ERFVIIs are destabilized, whereas an absence of either permits their accumulation; ERFVIIs therefore coordinate plant homeostatic responses to oxygen availability and control a wide range of NO-mediated processes. ERFVIIs have a variety of context-specific protein and gene interaction partners, and also modulate gibberellin and abscisic acid signaling to regulate diverse developmental processes and stress responses. This update discusses recent advances in our understanding of ERFVII regulation and function, highlighting their role as central regulators of gaseous signal transduction at the interface of ethylene, oxygen, and NO signaling.

Barley has two peroxisomal ABC transporters with multiple functions in ß-oxidation.

In oilseed plants, peroxisomal ß-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of ß-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 µM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid ß-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ.

The Cys-Arg/N-End Rule Pathway Is a General Sensor of Abiotic Stress in Flowering Plants.

Abiotic stresses impact negatively on plant growth, profoundly affecting yield and quality of crops. Although much is known about plant responses, very little is understood at the molecular level about the initial sensing of environmental stress. In plants, hypoxia (low oxygen, which occurs during flooding) is directly sensed by the Cys-Arg/N-end rule pathway of ubiquitin-mediated proteolysis, through oxygen-dependent degradation of group VII Ethylene Response Factor transcription factors (ERFVIIs) via amino-terminal (Nt-) cysteine [1, 2]. Using Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), we show that the pathway regulates plant responses to multiple abiotic stresses. In Arabidopsis, genetic analyses revealed that response to these stresses is controlled by N-end rule regulation of ERFVII function. Oxygen sensing via the Cys-Arg/N-end rule in higher eukaryotes is linked through a single mechanism to nitric oxide (NO) sensing [3, 4]. In plants, the major mechanism of NO synthesis is via NITRATE REDUCTASE (NR), an enzyme of nitrogen assimilation [5]. Here, we identify a negative relationship between NR activity and NO levels and stabilization of an artificial Nt-Cys substrate and ERFVII function in response to environmental changes. Furthermore, we show that ERFVIIs enhance abiotic stress responses via physical and genetic interactions with the chromatin-remodeling ATPase BRAHMA. We propose that plants sense multiple abiotic stresses through the Cys-Arg/N-end rule pathway either directly (via oxygen sensing) or indirectly (via NO sensing downstream of NR activity). This single mechanism can therefore integrate environment and response to enhance plant survival.