ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana.

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

A Yeast-Based Functional Assay to Study Plant N-Degron - N-Recognin Interactions.

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2.

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development.

Detection of Potential Chloroplastic Substrates for Polyphenol Oxidase Suggests a Role in Undamaged Leaves.

Polyphenol oxidases (PPOs) have a recognized role during pathogen and arthropod attack. As an immediate consequence of such wounding, cellular compartmentation is destroyed allowing the chloroplastic PPO enzyme to interact with vacuolar substrates catalyzing the oxidation of monophenols and/or o-diphenols to o-diquinones. This ultimately results in a reduction in the nutritional value of wounded tissue through the formation of non-digestible secondary melanin pigments. However, the chloroplastic location of PPO enzyme could indicate a role for PPO in undamaged tissues. In this study, a wild-type red clover population exhibiting high leaf PPO activity had significantly higher yield than a low leaf PPO mutant population while leaf isoflavonoids and hydroxycinnammates (PPO substrates) accumulated at similar levels in these plants. These data suggest that the presence of leaf PPO activity affects plant vigor. Understanding how this advantage is conferred requires knowledge of the cellular mechanism, including intra-organellar substrates. Here we present evidence of candidate PPO substrates within chloroplasts of wild-type red clover, including the monophenolic acid, coumaroyl malate, and low levels of the diphenolic acid, phaselic acid (caffeoyl malate). Interestingly, chloroplastic phaselic acid concentration increased significantly under certain growth conditions. We discuss the implications of this in regard to a potential role for chloroplastic PPO in undamaged leaves.

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency.

BACKGROUND AND AIMS: Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress. METHODS: Photosynthesis (A, Fv/Fm, ΦPSII, qN, qP, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense 'Milvus') under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m(2 )s(-1)) or 0-10 µm methyl viologen. Foliar protein content and oxidation state were also determined. KEY RESULTS: Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses. CONCLUSIONS: The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.

Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?

Polyphenol oxidase (PPO) catalyses the oxidation of monophenols and/or o-diphenols to o-quinones with the concomitant reduction of oxygen to water which results in protein complexing and the formation of brown melanin pigments. The most frequently suggested role for PPO in plants has been in defence against herbivores and pathogens, based on the physical separation of the chloroplast-localized enzyme from the vacuole-localized substrates. The o-quinone-protein complexes, formed as a consequence of cell damage, may reduce the nutritional value of the tissue and thereby reduce predation but can also participate in the formation of structural barriers against invading pathogens. However, since a sufficient level of compartmentation-based regulation could be accomplished if PPO was targeted to the cytosol, the benefit derived by some plant species in having PPO present in the chloroplast lumen remains an intriguing question. So is there more to the chloroplastic location of PPO? An interaction between PPO activity and photosynthesis has been proposed on more than one occasion but, to date, evidence either for or against direct involvement has been equivocal, and the lack of identified chloroplastic substrates remains an issue. Similarly, PPO has been suggested to have both pro- and anti-oxidant functions. Nevertheless, several independent lines of evidence suggest that PPO responds to environmental conditions and could be involved in the response of plants to abiotic stress. This review highlights our current understanding of the in vivo functions of PPO and considers the potential opportunities it presents for exploitation to increase stress tolerance in food crops.